Rna interference suppression of neurodegenerative diseases and methods of use thereof

ABSTRACT

The present invention is directed to small interfering RNA molecules (siRNA) targeted against nucleic acid sequence that encodes huntingtin or ataxin-1, and methods of using these siRNA molecules.

CLAIM OF PRIORITY

This patent application is a continuation application of U.S.application Ser. No. 11/597,225 filed on Nov. 21, 2006, which is aNational Stage application under 35 U.S.C. §371 and claims benefit under35 U.S.C. §119(a) of International Application No. PCT/US2005/019749having an International Filing Date of Jun. 2, 2005, which is acontinuation-in-part application of U.S. application Ser. No. 11/048,627filed on Jan. 31, 2005, which is a continuation-in-part application ofU.S. application Ser. No. 10/738,642 filed on Dec. 16, 2003, and is acontinuation-in-part application of U.S. application Ser. No. 10/859,751filed on Jun. 2, 2004, both of which are continuation-in-partapplications of International PCT Application No. PCT/US03/16887 filedon May 26, 2003, which is a continuation-in-part of application U.S.application Ser. No. 10/430,351 filed on May 5, 2003, which is acontinuation of U.S. application Ser. No. 10/322,086 filed on Dec. 17,2002, which is a continuation-in-part application of U.S. applicationSer. No. 10/212,322, filed Aug. 5, 2002. The instant application claimsthe benefit of all the listed applications, which are herebyincorporated by reference herein in their entireties, including thedrawings.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Work relating to this application was supported by grants from theNational Institutes of Health (NS044494, NS38712, HD44093, DK54759, andNS22920). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Double-stranded RNA (dsRNA) can induce sequence-specificposttranscriptional gene silencing in many organisms by a process knownas RNA interference (RNAi). However, in mammalian cells, dsRNA that is30 base pairs or longer can induce sequence-nonspecific responses thattrigger a shut-down of protein synthesis. RNA fragments are thesequence-specific mediators of RNAi. Interference of gene expression bythese small interfering RNA (siRNA) is now recognized as a naturallyoccurring strategy for silencing genes in C. elegans, Drosophila,plants, and in mouse embryonic stem cells, oocytes and early embryos.

SUMMARY OF THE INVENTION

The dominant polyglutamine expansion diseases, which includeSpinocerebellar ataxia type 1 (SCA1) and Huntington's disease (HD), areprogressive, untreatable neurodegenerative disorders. In inducible mousemodels of SCA1 and HD, repression of mutant allele expression improvesdisease phenotypes. Thus, therapies designed to inhibit disease geneexpression would be beneficial. In this study, the ability of RNAinterference (RNAi) to inhibit polyglutamine-induced neurodegenerationcaused by mutant ataxin-1 was evaluated in a mouse model of SCA1. Uponintracerebellar injection, recombinant AAV vectors expressing shRNAsprofoundly improved motor coordination, restored cerebellar morphology,and resolved characteristic ataxin-1 inclusions in Purkinje cells ofSCA1 mice. The present invention provides methods of using RNAi in vivoto treat dominant neurodegenerative diseases. “Treating” as used hereinrefers to ameliorating at least one symptom of, curing and/or preventingthe development of a disease or a condition.

In certain embodiment of the invention, siRNAs are employed to inhibitexpression of a target gene. By “inhibit expression” is meant to reduce,diminish or suppress expression of a target gene. Expression of a targetgene may be inhibited via “gene silencing.” Gene silencing refers to thesuppression of gene expression, e.g., transgene, heterologous geneand/or endogenous gene expression, which may be mediated throughprocesses that affect transcription and/or through processes that affectpost-transcriptional mechanisms. In some embodiments, gene silencingoccurs when siRNA initiates the degradation of the mRNA transcribed froma gene of interest in a sequence-specific manner via RNA interference,thereby preventing translation of the gene's product (for a review, seeBrantl, 2002).

The present invention provides an isolated RNA duplex that has a firststrand of RNA and a second strand of RNA, wherein the first strand hasat least 15 contiguous nucleotides encoded by shSCA1.F10 (SEQ ID NO:102)or shSCA1.F11 (SEQ ID NO:103), and wherein the second strand iscomplementary to at least 12 contiguous nucleotides of the first strand.In one embodiment, the first strand of RNA is encoded by shSCA1.F10 orby shSCA1.F11. As used herein the term “encoded by” is used in a broadsense, similar to the term “comprising” in patent terminology. Forexample, the statement “the first strand of RNA is encoded by SEQ IDNO:102” means that the first strand of RNA sequence corresponds to theRNA sequence transcribed from the DNA sequence indicated in SEQ IDNO:102, but may also contain additional nucleotides at either the 3′ endor at the 5′ end of the RNA molecule.

The present invention also provides an RNA duplex (under physiologicalconditions) having a first strand of RNA and a second strand of RNA,wherein the first strand has at least 15 contiguous nucleotides encodedby (a) shHDEx2.1 (5′-AAGAAAGAACTTTCAGCTACC-3′, SEQ ID NO:96)), (b)shHDEx2.2 19 nt (5′-AGAACTTTCAGCTACCAAG-3′ (SEQ ID NO:97)), (c)shHDEx2.2 21 nt (5′-AAAGAACTTTCAGCTACCAAG-3′ (SEQ ID NO:98)), (d)shHDEx3.1 19 nt (5′-TGCCTCAACAAAGTTATCA-3′ (SEQ ID NO:99)), or (e)shHDEx3.1 21 nt (5′-AATGCCTCAACAAAGTTATCA-3′ (SEQ ID NO:100)), (f)siEX58#1 (5′-GAGGAAGAGGAGGAGGCCGAC-3′ (SEQ ID NO:101)), or (g) siEX58#2(5′-AAGAGGAGGAGGCCGACGCCC-3′ (SEQ ID NO:90)) and wherein the secondstrand is complementary to at least 12 contiguous nucleotides of thefirst strand. In further embodiments, the first strand has at least 15contiguous nucleotides encoded by SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:63, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86, or SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:96 throughSEQ ID NO:101 or SEQ ID NO:106 through SEQ ID NO:142, and wherein thesecond strand is complementary to at least 12 contiguous nucleotides ofthe first strand. In one embodiment, the loop structure corresponds toSEQ ID NO:58. In one embodiment, the first strand corresponds to SEQ IDNO:56 and the second strand corresponds to SEQ ID NO:57.

The reference to siRNAs herein is meant to include shRNAs and othersmall RNAs that can or are capable of modulating the expression of HDgene, for example via RNA interference. Such small RNAs include withoutlimitation, shRNAs and microRNAs (miRNAs).

In certain embodiments, the RNA duplex described above is between 15 and30 base pairs in length, such as 19 or 21 base pairs in length. Incertain embodiments, the first and/or second strand further comprises anoverhang, such as a 3′ overhang region, a 5′ overhang region, or both 3′and 5′ overhang regions. The two strands of RNA in the siRNA may becompletely complementary, or one or the other of the strands may have an“overhang region” (i.e., a portion of the RNA that does not bind withthe second strand). Such an overhang region may be from 1 to 10nucleotides in length.

In certain embodiments, in the RNA duplex described above, the firststrand and the second strand are operably linked by means of an RNA loopstrand to form a hairpin structure to form a duplex structure and a loopstructure. In certain embodiments, the loop structure contains from 4 to10 nucleotides, such as 4, 5 or 6 nucleotides. In certain embodiments,the loop structure corresponds to SEQ ID NO:61 or SEQ ID NO:64.

The present invention further provides expression cassettes containing anucleic acid encoding at least one strand of the RNA duplex describedabove. The expression cassette may further contain a promoter, such as aregulatable promoter or a constitutive promoter. Examples of suitablepromoters include a CMV, RSV, pol II or pol III promoter. The expressioncassette may further contain a polyadenylation signal (such as asynthetic minimal polyadenylation signal) and/or a marker gene.

The present invention also provides vectors containing the expressioncassettes described above. Examples of appropriate vectors includeadenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV,or murine Maloney-based viral vectors. In one embodiment, the vector isan adenoviral vector. In certain embodiments, a vector may contain twoexpression cassettes, a first expression cassette containing a nucleicacid encoding the first strand of the RNA duplex and a second expressioncassette containing a nucleic acid encoding the second strand of the RNAduplex.

The present invention provides cells (such as a mammalian cell)containing the expression cassette or vectors described above. Thepresent invention also provides a non-human mammal containing theexpression cassette or vectors described above.

The present invention provides a method of suppressing the accumulationof huntingtin or ataxin-1 in a cell by introducing a ribonucleic acid(RNA) described above into the cell in an amount sufficient to suppressaccumulation of huntingtin or ataxin-1 in the cell. In certainembodiments, the accumulation of huntingtin or ataxin-1 is suppressed byat least 10%. The accumulation of huntingtin or ataxin-1 is suppressedby at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99%.

The present invention provides a method of preventing cytotoxic effectsof mutant huntingtin or ataxin-1 in a cell by introducing a ribonucleicacid (RNA) described above into the cell in an amount sufficient tosuppress accumulation of huntingtin or ataxin-1, and wherein the RNAprevents cytotoxic effects of huntingtin or ataxin-1 in the oculartissue cell.

The present invention provides a method to inhibit expression of ahuntingtin or ataxin-1 gene in a cell by introducing a ribonucleic acid(RNA) described above into the cell in an amount sufficient to inhibitexpression of the huntingtin or ataxin-1, and wherein the RNA inhibitsexpression of the huntingtin or ataxin-1 gene. The huntingtin orataxin-1 is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% 95%, or 99%.

The present invention provides a method to inhibit expression of ahuntingtin or ataxin-1 gene in a mammal (e.g., a human) by (a) providinga mammal containing a neuronal cell, wherein the neuronal cell containsthe huntingtin or ataxin-1 gene and the neuronal cell is susceptible toRNA interference, and the huntingtin or ataxin-1 gene is expressed inthe neuronal cell; and (b) contacting the mammal with a ribonucleic acid(RNA) or a vector described above, thereby inhibiting expression of thehuntingtin or ataxin-1 gene. In certain embodiments, the accumulation ofhuntingtin or ataxin-1 is suppressed by at least 10%. The huntingtin orataxin-1 is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% 95%, or 99%. In certain embodiments, the cell located in vivoin a mammal.

The present invention provides a viral vector comprising a promoter anda micro RNA (miRNA) shuttle containing an embedded siRNA specific for atarget sequence. In certain embodiments, the promoter is an induciblepromoter. In certain embodiments, the vector is an adenoviral,lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murineMaloney-based viral vector. In certain embodiments, the targetedsequence is a sequence associated with a condition amenable to siRNAtherapy, such as a neurodegenerative disease. An example ofneurodegenerative diseases is a trinucleotide-repeat disease, such as adisease associated with polyglutamine repeats. These diseases includeHuntington's disease or a spinocerebellar ataxia (SCA). Examples of SCAdiseases are SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17. The target sequenceof the present invention, in certain embodiments, is a sequence encodingataxin-1 or huntingtin.

The present invention provides a method of preventing cytotoxic effectsof neurodegenerative disease in a mammal in need thereof, by introducingthe vector encoding a miRNA described in the preceding paragraph into acell in an amount sufficient to suppress accumulation of a proteinassociated with the neurodegenerative disease, and wherein the RNAprevents cytotoxic effects of neurodegenerative disease.

The present invention also provides a method to inhibit expression of aprotein associated with the neurodegenerative disease in a mammal inneed thereof, by introducing the vector encoding a miRNA described aboveinto a cell in an amount sufficient to inhibit expression of the proteinassociated with the neurodegenerative disease, wherein the RNA inhibitsexpression of the protein associated with the neurodegenerative disease.The protein associated with the neurodegenerative disease is inhibitedby at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99%.

This invention relates to compounds, compositions, and methods usefulfor modulating Huntington's Disease (also referred to as huntingtin,htt, or HD) gene expression using short interfering nucleic acid (siRNA)molecules. This invention also relates to compounds, compositions, andmethods useful for modulating the expression and activity of other genesinvolved in pathways of HD gene expression and/or activity by RNAinterference (RNAi) using small nucleic acid molecules. In particular,the instant invention features small nucleic acid molecules, such asshort interfering nucleic acid (siRNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(sbRNA) molecules and methods used to modulate the expression HD genes.A siRNA of the instant invention can be chemically synthesized,expressed from a vector or enzymatically synthesized.

In one embodiment, the present invention provides an AAV-1 expressedsiRNA comprising an isolated first strand of RNA of 15 to 30 nucleotidesin length and an isolated second strand of RNA of 15 to 30 nucleotidesin length, wherein the first or second strand comprises a sequence thatis complementary to a nucleotide sequence encoding a mutant Huntington'sDisease protein, wherein at least 12 nucleotides of the first and secondstrands are complementary to each other and form a small interfering RNA(siRNA) duplex under physiological conditions, and wherein the siRNAsilences the expression of the nucleotide sequence encoding the mutantHuntington's Disease protein in the cell. In one embodiment, the firstor second strand comprises a sequence that is complementary to both amutant and wild-type Huntington's disease allele, and the siRNA silencesthe expression of the nucleotide sequence encoding the mutantHuntington's Disease protein and wild-type Huntington's Disease proteinin the cell.

In one embodiment, the present invention provides an AAV-1 expressedsiRNA comprising an isolated first strand of RNA of 15 to 30 nucleotidesin length and an isolated second strand of RNA of 15 to 30 nucleotidesin length, wherein the first or second strand comprises a sequence thatis complementary to both a nucleotide sequence encoding a wild-type andmutant Huntington's Disease protein, wherein at least 12 nucleotides ofthe first and second strands are complementary to each other and form asmall interfering RNA (siRNA) duplex under physiological conditions, andwherein the siRNA silences the expression of the nucleotide sequenceencoding the wild-type and mutant Huntington's Disease protein in thecell. In one embodiment, an AAV-1 vector of the invention is apsuedotyped rAAV-1 vector.

In one embodiment, the present invention provides a mammalian cellcontaining an isolated first strand of RNA for example corresponding toSEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80,SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, or SEQ ID NO:88, and anisolated second strand of RNA of 15 to 30 nucleotides in length, whereinthe first strand contains a sequence that is complementary to anucleotide sequence encoding a Huntington's Disease protein (htt), suchas wherein at least 12 nucleotides of the first and second strands arecomplementary to each other and form a small interfering RNA (siRNA)duplex for example under physiological conditions, and wherein the siRNAsilences the expression of the Huntington's Disease (HD) gene in thecell, for example by targeting the cleavage of RNA encoded by the HDgene or via translational blocking of the HD gene expression. SEQ IDNO:60 through SEQ ID NO:89 are all represented herein as DNA sequences.However, as used herein when a claim indicates an RNA “corresponding to”it is meant the RNA that has the same sequence as the DNA, except thaturacil is substituted for thymine. For example, SEQ ID NO:61 is5′-GAAGCTTG-3′, and the RNA corresponding to this sequence is5′-GAAGCUUG-3′ (SEQ ID NO: 58).

The present invention also provides a mammalian cell containing anexpression cassette encoding an isolated first strand of RNAcorresponding to, for example, SEQ ID NO:56 or SEQ ID NO:57, andencoding an isolated second strand of RNA of 15 to 30 nucleotides inlength, wherein the first or second strand comprises a sequence that iscomplementary to a nucleotide sequence encoding a Huntington's Diseaseprotein (htt), for example wherein at least 12 nucleotides of the firstand second strands are complementary to each other and form a smallinterfering RNA (siRNA) duplex for example under physiologicalconditions, and wherein the siRNA silences the expression of theHuntington's Disease gene in the cell, for instance by targeting thecleavage of RNA encoded by the HD gene or via translational blocking ofthe HD gene expression. The expression cassette may further include apromoter, such as a regulatable promoter or a constitutive promoter.Examples of suitable promoters include without limitation a pol IIpromoter such as cytomegalovirus (CMV), Rous Sarcoma Virus (RSV), polIII promoters such as U6, and any other pol II or pol III promoter as isknown in the art. The expression cassette may further optionally includea marker gene, such as a stuffer fragment comprising a marker gene. Theexpression cassette may be contained in a vector, such as an adenoviral,lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murineMaloney-based viral vector. In one embodiment, the first strandcorresponds to SEQ ID NO:56 and the second strand corresponds to SEQ IDNO:57.

The present invention provides a small interfering RNA (siRNA)containing a first strand of RNA corresponding to for example SEQ IDNO:56 or SEQ ID NO:57, and a second strand of RNA of 15 to 30nucleotides in length, wherein the first or second strand comprises asequence that is complementary to a nucleotide sequence encoding aHuntington's Disease protein (htt), for example wherein at least 12nucleotides of the first and second strands are complementary to eachother and form an siRNA duplex under physiological conditions, whereinthe duplex is between 15 and 30 base pairs in length, and wherein thesiRNA silences the expression of the Huntington's Disease gene in thecell, for instance via RNA interference.

The present invention provides a method of performing Huntington'sDisease gene silencing in a mammal by administering to the mammal anexpression cassette encoding an isolated first strand of RNAcorresponding to for example SEQ ID NO:56 or SEQ ID NO:57, and encodingan isolated second strand of RNA of 15 to 30 nucleotides in length,wherein the first or second strand comprises a sequence that iscomplementary to a nucleotide sequence encoding a Huntington's Diseaseprotein (htt), for example wherein at least 12 nucleotides of the firstand second strands are complementary to each other and form a smallinterfering RNA (siRNA) duplex under physiological conditions, andwherein the expression of the siRNA from the expression cassettesilences the expression of the Huntington's Disease gene in the mammal,for instance via RNA interference.

The present invention provides an isolated RNA comprising for exampleSEQ ID NO:59 that functions in RNA interference to a sequence encoding amutant Huntington's Disease protein (htt).

The present invention provides an isolated RNA duplex comprising a firststrand of RNA corresponding to for example SEQ ID NO:56 and a secondstrand of RNA corresponding to for example by SEQ ID NO:57. The firstand/or second strand optionally further include a 3′ overhang region, a5′ overhang region, or both 3′ and 5′ overhang regions, and the overhangregion (or regions) can be from 1 to 10 nucleotides in length. Further,the first strand and the second strand can be operably linked by meansof an RNA loop strand to form a hairpin structure comprising a duplexstructure and a loop structure. This loop structure, if present may befrom 4 to 10 nucleotides. In one embodiment, the loop structurecorresponds to SEQ ID NO:58 or a portion thereof.

The present invention provides a vector, such as an AAV vector,comprising two expression cassettes, a first expression cassettecomprising a nucleic acid encoding the first strand of the RNA duplexcorresponding to for example SEQ ID NO:56 and a second expressioncassette comprising a nucleic acid encoding the second strand of the RNAduplex corresponding to for example SEQ ID NO:57. The present inventionalso provides a cell containing this vector. In one embodiment, the cellis a mammalian cell.

The present invention provides a mammalian cell containing an isolatedfirst strand of RNA of 15 to 30 nucleotides in length, and an isolatedsecond strand of RNA of 15 to 30 nucleotides in length, wherein thefirst strand contains a sequence that is complementary to for example atleast 15 nucleotides of RNA encoded by a targeted gene of interest (forexample the HD gene), wherein for example at least 12 nucleotides of thefirst and second strands are complementary to each other and form asmall interfering RNA (siRNA) duplex for example under physiologicalconditions, and wherein the siRNA silences (for example via RNAinterference) only one allele of the targeted gene (for example themutant allele of HD gene) in the cell. The duplex of the siRNA may bebetween 15 and 30 base pairs in length. The two strands of RNA in thesiRNA may be completely complementary, or one or the other of thestrands may have an “overhang region” or a “bulge region” (i.e., aportion of the RNA that does not bind with the second strand or where aportion of the RNA sequence is not complementary to the sequence of theother strand). These overhangs may be at the 3′ end or at the 5′ region,or at both 3′ and 5′ ends. Such overhang regions may be from 1 to 10(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or more nucleotides in length. Thebulge regions may be at the ends or in the internal regions of the siRNAduplex. Such bulge regions may be from 1-5 (e.g., 1, 2, 3, 4, 5) or morenucleotides long. Such bulge regions may be the bulge regionscharacteristics of miRNAs. In the present invention, the first andsecond strand of RNA may be operably linked together by means of an RNAloop strand to form a hairpin structure to form a “duplex structure” anda “loop structure.” These loop structures may be from 4 to 10 (e.g., 4,5, 6, 7, 8, 9, 10) or more nucleotides in length. For example, the loopstructure may be 4, 5 or 6 nucleotides long.

The present invention also provides a mammalian cell that contains anexpression cassette encoding an isolated first strand of RNA of 15 to 30nucleotides in length, and an isolated second strand of RNA of 15 to 30nucleotides in length, wherein the first strand contains a sequence thatis complementary to for example at least 15 contiguous nucleotides ofRNA encoded by a targeted gene of interest (for example the HD gene),wherein for example at least 12 nucleotides of the first and secondstrands are complementary to each other and form a small interfering RNA(siRNA) duplex, for example under physiological conditions, and whereinthe siRNA silences (for example via RNA interference) only one allele ofthe targeted gene (for example the mutant allele of HD gene) in thecell. These expression cassettes may further contain a promoter. Suchpromoters can be regulatable promoters or constitutive promoters.Examples of suitable promoters include a CMV, RSV, pol II or pol IIIpromoter. The expression cassette may further contain a polyadenylationsignal, such as a synthetic minimal polyadenylation signal. Theexpression cassette may further contain a marker gene. The expressioncassette may be contained in a vector. Examples of appropriate vectorsinclude adenoviral, lentiviral, adeno-associated viral (AAV),poliovirus, HSV, or murine Maloney-based viral vectors. In oneembodiment, the vector is an adenoviral vector or an adeno-associatedviral vector.

In the present invention, the alleles of the targeted gene may differ byseven or fewer nucleotides (e.g., 7, 6, 5, 4, 3, 2 or 1 nucleotides).For example the alleles may differ by only one nucleotide. Examples oftargeted gene transcripts include transcripts encoding abeta-glucuronidase, TorsinA, Ataxin-3, Tau, or huntingtin. The targetedgenes and gene products (i.e., a transcript or protein) may be fromdifferent species of organisms, such as a mouse allele or a human alleleof a target gene.

The present invention also provides an isolated RNA duplex containing afirst strand of RNA and a second strand of RNA, wherein the first strandcontains for example at least 15 nucleotides complementary to mutantTorsinA represented for example by SEQ ID NO:55(5′-GTAAGCAGAGTGGCTGAGATGACATTTTTCCCCAAAGAG-3′), and wherein the secondstrand is complementary to for example at least 12 contiguousnucleotides of the first strand. In one embodiment of the invention(mutA-si), the first strand of RNA corresponds to for example SEQ IDNO:49 and the second strand of RNA corresponds to for example SEQ IDNO:50. In an alternative embodiment (mutB-si), the first strand of RNAcorresponds to for example SEQ ID NO:51 and the second strand of RNAcorresponds to for example SEQ ID NO:52. In another embodiment(mutC-si), the first strand of RNA corresponds to for example SEQ IDNO:53 and second strand of RNA corresponds to for example SEQ ID NO:54.As used herein the term “encoded by” means that the DNA sequence istranscribed into the RNA of interest. This term is used in a broadsense, similar to the term “comprising” in patent terminology. Forexample, the statement “the first strand of RNA is encoded by SEQ IDNO:49” means that the first strand of RNA sequence corresponds to theDNA sequence indicated in SEQ ID NO:49, but may also contain additionalnucleotides at either the 3′ end or at the 5′ end of the RNA molecule.

The present invention further provides an RNA duplex containing a firststrand of RNA and a second strand of RNA, wherein the first strandcontains for example at least 15 contiguous nucleotides complementary tomutant Ataxin-3 transcript encoded by SEQ ID NO:8, and wherein thesecond strand is complementary to for example at least 12 contiguousnucleotides of the first strand. In one embodiment (siC7/8), the firststrand of RNA is encoded by SEQ ID NO:19 and the second strand of RNA isencoded by SEQ ID NO: 20. In another embodiment (siC10), the firststrand of RNA is encoded by SEQ ID NO:21 and the second strand of RNA isencoded by SEQ ID NO:22.

The present invention further provides an RNA duplex containing a firststrand of RNA and a second strand of RNA, wherein the first strandcontains for example at least 15 contiguous nucleotides complementary tomutant Tau transcript for example encoded by SEQ ID NO:39 (siA9/C12),and wherein the second strand is complementary to at least 12 contiguousnucleotides of the first strand. The second strand may be encoded forexample by SEQ ID NO:40.

The RNA duplexes of the present invention are between 15 and 30 basepairs in length. For example they may be between 19 and 25 base pairs inlength or 19-27 base-pairs in length. As discussed above the firstand/or second strand further may optionally comprise an overhang region.These overhangs may be at the 3′ end or at the 5′ overhang region, or atboth 3′ and 5′ ends. Such overhang regions may be from 1 to 10nucleotides in length. The RNA duplex of the present invention mayoptionally include nucleotide bulge regions. The bulge regions may be atthe ends or in the internal regions of the siRNA duplex. Such bulgeregions may be from 1-5 nucleotides long. Such bulge regions may be thebulge regions characteristics of miRNAs. In the present invention, thefirst and second strand of RNA may be operably linked together by meansof an RNA loop strand to form a hairpin structure to form a “duplexstructure” and a “loop structure.” These loop structures may be from 4to 10 nucleotides in length. For example, the loop structure may be 4, 5or 6 nucleotides long.

In the present invention, an expression cassette may contain a nucleicacid encoding at least one strand of the RNA duplex described above.Such an expression cassette may further contain a promoter. Theexpression cassette may be contained in a vector. These cassettes andvectors may be contained in a cell, such as a mammalian cell. Anon-human mammal may contain the cassette or vector. The vector maycontain two expression cassettes, the first expression cassettecontaining a nucleic acid encoding the first strand of the RNA duplex,and a second expression cassette containing a nucleic acid encoding thesecond strand of the RNA duplex.

In one embodiment, the present invention further provides a method ofperforming gene silencing in a mammal or mammalian cell by administeringto the mammal an isolated first strand of RNA of about 15 to about 30nucleotides (for example 19-27 nucleotides) in length, and an isolatedsecond strand of RNA of 15 to 30 nucleotides (for example 19-27nucleotides) in length, wherein the first strand contains for example atleast 15 contiguous nucleotides complementary to a targeted gene ofinterest (such as HD gene), wherein for example at least 12 nucleotidesof the first and second strands are complementary to each other and forma small interfering RNA (siRNA) duplex for example under physiologicalconditions, and wherein the siRNA silences only one or both alleles ofthe targeted gene (for example the wild type and mutant alleles of HDgene) in the mammal or mammalian cell. In one example, the gene is abeta-glucuronidase gene. The alleles may be murine-specific andhuman-specific alleles of beta-glucuronidase. Examples of genetranscripts include an RNA transcript complementary to TorsinA,Ataxin-3, huntingtin or Tau. The targeted gene may be a gene associatedwith a condition amenable to siRNA therapy. For example, the conditionamenable to siRNA therapy could be a disabling neurological disorder.

“Neurological disease” and “neurological disorder” refer to bothhereditary and sporadic conditions that are characterized by nervoussystem dysfunction, and which may be associated with atrophy of theaffected central or peripheral nervous system structures, or loss offunction without atrophy. A neurological disease or disorder thatresults in atrophy is commonly called a “neurodegenerative disease” or“neurodegenerative disorder.” Neurodegenerative diseases and disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, and repeat expansion neurodegenerativediseases, e.g., diseases associated with expansions of trinucleotiderepeats such as polyglutamine (polyQ) repeat diseases, e.g.,Huntington's disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,SCA6, SCA7, and SCA17), spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA). An example of a disablingneurological disorder that does not appear to result in atrophy is DYT1dystonia. The gene of interest may encode a ligand for a chemokineinvolved in the migration of a cancer cell, or a chemokine receptor.

The present invention further provides a method of substantiallysilencing a target gene of interest or targeted allele for the gene ofinterest in order to provide a therapeutic effect. As used herein theterm “substantially silencing” or “substantially silenced” refers todecreasing, reducing, or inhibiting the expression of the target gene ortarget allele by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% to 100%. As used herein theterm “therapeutic effect” refers to a change in the associatedabnormalities of the disease state, including pathological andbehavioral deficits; a change in the time to progression of the diseasestate; a reduction, lessening, or alteration of a symptom of thedisease; or an improvement in the quality of life of the personafflicted with the disease. Therapeutic effect can be measuredquantitatively by a physician or qualitatively by a patient afflictedwith the disease state targeted by the siRNA. In certain embodimentswherein both the mutant and wild type allele are substantially silenced,the term therapeutic effect defines a condition in which silencing ofthe wild type allele's expression does not have a deleterious or harmfuleffect on normal functions such that the patient would not have atherapeutic effect.

In one embodiment, the present invention further provides a method ofperforming allele-specific gene silencing in a mammal by administeringto the mammal an isolated first strand of RNA of 15 to 30 nucleotides inlength, and an isolated second strand of RNA of 15 to 30 nucleotides inlength, wherein the first strand contains for example at least 15contiguous nucleotides complementary to a targeted gene of interest,wherein for example at least 12 nucleotides of the first and secondstrands are complementary to each other and form a small interfering RNA(siRNA) duplex for example under physiological conditions, and whereinthe siRNA silences only one allele of the targeted gene in the mammal.The alleles of the gene may differ by seven or fewer base pairs, such asby only one base pair. In one example, the gene is a beta-glucuronidasegene. The alleles may be murine-specific and human-specific alleles ofbeta-glucuronidase. Examples of gene transcripts include an RNAtranscript complementary to TorsinA, Ataxin-3, huntingtin or Tau. Thetargeted gene may be a gene associated with a condition amenable tosiRNA therapy. For example, the condition amenable to siRNA therapycould be a disabling neurological disorder.

“Neurological disease” and “neurological disorder” refer to bothhereditary and sporadic conditions that are characterized by nervoussystem dysfunction, and which may be associated with atrophy of theaffected central or peripheral nervous system structures, or loss offunction without atrophy. A neurological disease or disorder thatresults in atrophy is commonly called a “neurodegenerative disease” or“neurodegenerative disorder.” Neurodegenerative diseases and disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, and repeat expansion neurodegenerativediseases, e.g., diseases associated with expansions of trinucleotiderepeats such as polyglutamine (polyQ) repeat diseases, e.g.,Huntington's disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,SCA6, SCA7, and SCA17), spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA). An example of a disablingneurological disorder that does not appear to result in atrophy is DYT1dystonia. The gene of interest may encode a ligand for a chemokineinvolved in the migration of a cancer cell, or a chemokine receptor.

In one embodiment, the present invention further provides a method ofsubstantially silencing both alleles (e.g., both mutant and wild typealleles) of a target gene. In certain embodiments, the targeting of bothalleles of a gene target of interest can confer a therapeutic effect byallowing a certain level of continued expression of the wild-type allelewhile at the same time inhibiting expression of the mutant (e.g.,disease associated) allele at a level that provides a therapeuticeffect. For example, a therapeutic effect can be achieved by conferringon the cell the ability to express siRNA as an expression cassette,wherein the expression cassette contains a nucleic acid encoding a smallinterfering RNA molecule (siRNA) targeted against both alleles, andwherein the expression of the targeted alleles are silenced at a levelthat inhibits, reduces, or prevents the deleterious gain of functionconferred by the mutant allele, but that still allows for adequateexpression of the wild type allele at a level that maintains thefunction of the wild type allele. Examples of such wild type and mutantalleles include without limitation those associated with polyglutaminediseases such as Huntington's Disease.

In one embodiment, the present invention further provides a method ofsubstantially silencing a target allele while allowing expression of awild-type allele by conferring on the cell the ability to express siRNAas an expression cassette, wherein the expression cassette contains anucleic acid encoding a small interfering RNA molecule (siRNA) targetedagainst a target allele, wherein expression from the targeted allele issubstantially silenced but wherein expression of the wild-type allele isnot substantially silenced.

In one embodiment, the present invention provides a method of treating adominantly inherited disease in an allele-specific manner byadministering to a patient in need thereof an expression cassette,wherein the expression cassette contains a nucleic acid encoding a smallinterfering RNA molecule (siRNA) targeted against a target allele,wherein expression from the target allele is substantially silenced butwherein expression of the wild-type allele is not substantiallysilenced.

In one embodiment, the present invention provides a method of treating adominantly inherited disease by administering to a patient in needthereof an expression cassette, wherein the expression cassette containsa nucleic acid encoding a small interfering RNA molecule (siRNA)targeted against both the mutant allele and the wild type allele of thetarget gene, wherein expression from the mutant allele is substantiallysilenced at a level that still allows for expression from the wild typeallele to maintain its function in the patient.

In one embodiment, the present invention also provides a method ofperforming allele-specific gene silencing by administering an expressioncassette containing a pol II promoter operably-linked to a nucleic acidencoding at least one strand of a small interfering RNA molecule (siRNA)targeted against a gene of interest, wherein the siRNA silences only oneallele of a gene.

In one embodiment, the present invention also provides a method ofperforming gene silencing by administering an expression cassettecontaining a pol II promoter operably-linked to a nucleic acid encodingat least one strand of a small interfering RNA molecule (siRNA) targetedagainst a gene of interest, wherein the siRNA silences one or bothalleles of the gene.

In one embodiment, the present invention provides a method of performingallele-specific gene silencing in a mammal by administering to themammal a vector containing an expression cassette, wherein theexpression cassette contains a nucleic acid encoding at least one strandof a small interfering RNA molecule (siRNA) targeted against a gene ofinterest, wherein the siRNA silences only one allele of a gene.

In one embodiment, the present invention provides a method of performinggene silencing in a mammal by administering to the mammal a vectorcontaining an expression cassette, wherein the expression cassettecontains a nucleic acid encoding at least one strand of a smallinterfering RNA molecule (siRNA) targeted against a gene of interest,wherein the siRNA silences one or both alleles of the gene.

In one embodiment, the present invention provides a method of screeningof allele-specific siRNA duplexes, involving contacting a cellcontaining a predetermined mutant allele with an siRNA with a knownsequence, contacting a cell containing a wild-type allele with an siRNAwith a known sequence, and determining if the mutant allele issubstantially silenced while the wild-type allele retains substantiallynormal activity.

In one embodiment, the present invention provides a method of screeningof specific siRNA duplexes, involving contacting a cell containing botha predetermined mutant allele and a predetermined wild-type allele withan siRNA with a known sequence, and determining if the mutant allele issubstantially silenced at a level that allows the wild-type allele toretain substantially normal activity.

In one embodiment, the present invention also provides a method ofscreening of allele-specific siRNA duplexes involving contacting a cellcontaining a predetermined mutant allele and a wild-type allele with ansiRNA with a known sequence, and determining if the mutant allele issubstantially silenced while the wild-type allele retains substantiallynormal activity.

In one embodiment, the present invention also provides a method fordetermining the function of an allele by contacting a cell containing apredetermined allele with an siRNA with a known sequence, anddetermining if the function of the allele is substantially modified.

In one embodiment, the present invention further provides a method fordetermining the function of an allele by contacting a cell containing apredetermined mutant allele and a wild-type allele with an siRNA with aknown sequence, and determining if the function of the allele issubstantially modified while the wild-type allele retains substantiallynormal function.

In one embodiment, the invention features a method for treating orpreventing Huntington's Disease in a subject or organism comprisingcontacting the subject or organism with a siRNA of the invention underconditions suitable to modulate the expression of the HD gene in thesubject or organism whereby the treatment or prevention of Huntington'sDisease can be achieved. In one embodiment, the HD gene target comprisesa mutant HD allele (e.g., an allele comprising a trinucleotide (CAG)repeat expansion). In one embodiment, the HD gene target comprises bothHD allele (e.g. an allele comprising a trinucleotide (CAG) repeatexpansion and a wild type allele). The siRNA molecule of the inventioncan be expressed from vectors as described herein or otherwise known inthe art to target appropriate tissues or cells in the subject ororganism.

In one embodiment, the invention features a method for treating orpreventing Huntington's Disease in a subject or organism comprising,contacting the subject or organism with a siRNA molecule of theinvention via local administration to relevant tissues or cells, such asbrain cells and tissues (e.g., basal ganglia, striatum, or cortex), forexample, by administration of vectors or expression cassettes of theinvention that provide siRNA molecules of the invention to relevantcells (e.g., basal ganglia, striatum, or cortex). In one embodiment, thesiRNA, vector, or expression cassette is administered to the subject ororganism by stereotactic or convection enhanced delivery to the brain.For example, U.S. Pat. No. 5,720,720 provides methods and devices usefulfor stereotactic and convection enhanced delivery of reagents to thebrain. Such methods and devices can be readily used for the delivery ofsiRNAs, vectors, or expression cassettes of the invention to a subjector organism, and is incorporated by reference herein in its entirety. USPatent Application Nos. 2002/0141980; 2002/0114780; and 2002/0187127 allprovide methods and devices useful for stereotactic and convectionenhanced delivery of reagents that can be readily adapted for deliveryof siRNAs, vectors, or expression cassettes of the invention to asubject or organism, and are incorporated by reference herein in theirentirety. Particular devices that may be useful in delivering siRNAs,vectors, or expression cassettes of the invention to a subject ororganism are for example described in US Patent Application No.2004/0162255, which is incorporated by reference herein in its entirety.The siRNA molecule of the invention can be expressed from vectors asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism.

In one embodiment, a viral vector of the invention is an AAV vector. An“AAV” vector refers to an adeno-associated virus, and may be used torefer to the naturally occurring wild-type virus itself or derivativesthereof. The term covers all subtypes, serotypes and pseudotypes, andboth naturally occurring and recombinant forms, except where requiredotherwise. As used herein, the term “serotype” refers to an AAV which isidentified by and distinguished from other AAVs based on capsid proteinreactivity with defined antisera, e.g., there are eight known serotypesof primate AAVs, AAV-1 to AAV-8. For example, serotype AAV-2 is used torefer to an AAV which contains capsid proteins encoded from the cap geneof AAV-2 and a genome containing 5′ and 3′ ITR sequences from the sameAAV-2 serotype. Pseudotyped AAV refers to an AAV that contains capsidproteins from one serotype and a viral genome including 5′-3′ ITRs of asecond serotype. Pseudotyped rAAV would be expected to have cell surfacebinding properties of the capsid serotype and genetic propertiesconsistent with the ITR serotype. Pseudotyped rAAV are produced usingstandard techniques described in the art. As used herein, for example,rAAV1 may be used to refer an AAV having both capsid proteins and 5′-3′ITRs from the same serotype or it may refer to an AAV having capsidproteins from serotype 1 and 5′-3′ ITRs from a different AAV serotype,e.g., AAV serotype 2. For each example illustrated herein thedescription of the vector design and production describes the serotypeof the capsid and 5′-3′ ITR sequences. The abbreviation “rAAV” refers torecombinant adeno-associated virus, also referred to as a recombinantAAV vector (or “rAAV vector”).

An “AAV virus” or “AAV viral particle” refers to a viral particlecomposed of at least one AAV capsid protein (preferably by all of thecapsid proteins of a wild-type AAV) and an encapsidated polynucleotide.If the particle comprises heterologous polynucleotide (i.e., apolynucleotide other than a wild-type AAV genome such as a transgene tobe delivered to a mammalian cell), it is typically referred to as“rAAV”.

In one embodiment, the AAV expression vectors are constructed usingknown techniques to at least provide as operatively linked components inthe direction of transcription, control elements including atranscriptional initiation region, the DNA of interest and atranscriptional termination region. The control elements are selected tobe functional in a mammalian cell. The resulting construct whichcontains the operatively linked components is flanked (5′ and 3′) withfunctional AAV ITR sequences.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome.

The nucleotide sequences of AAV ITR regions are known. See for exampleKotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I.“Parvoviridae and their Replication” in Fundamental Virology, 2ndEdition, (B. N. Fields and D. M. Knipe, eds.). As used herein, an “AAVITR” need not have the wild-type nucleotide sequence depicted, but maybe altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flanka selected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the heterologous sequence into the recipient cell genomewhen AAV Rep gene products are present in the cell.

In one embodiment, AAV ITRs can be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flank a selectednucleotide sequence in an AAV expression vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the DNA molecule into the recipient cell genome when AAVRep gene products are present in the cell.

In one embodiment, AAV capsids can be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAV6, or AAV8, and the AAV ITRS are derived form AAV serotype 2.Suitable DNA molecules for use in AAV vectors will be less than about 5kilobases (kb), less than about 4.5 kb, less than about 4 kb, less thanabout 3.5 kb, less than about 3 kb, less than about 2.5 kb in size andare known in the art Dong, J.-Y. et al. (Nov. 10, 1996). “QuantitativeAnalysis of the Packaging Capacity of Recombinant Adeno-AssociatedVirus,” Human Gene Ther. 7(17):2101-2112 and U.S. Pat. No. 6,596,535herein incorporated in its entirety. In some embodiments of theinvention the DNA molecules for use in the AAV vectors will containmultiple copies of the identical siRNA sequence. As used herein the termmultiple copies of an siRNA sequences means at least 2 copies, at least3 copies, at least 4 copies, at least 5 copies, at least 6 copies, atleast 7 copies, at least 8 copies, at least 9 copies, and at least 10copies. In some embodiments the DNA molecules for use in the AAV vectorswill contain multiple siRNA sequences. As used herein the termmultiple=Si RNA sequences means at least 2 siRNA sequences, at least 3siRNA sequences, at least 4 siRNA sequences, at least 5 siRNA sequences,at least 6 siRNA sequences, at least 7 siRNA sequences, at least 8 siRNAsequences, at least 9 siRNA sequences, and at least 10 siRNA sequences.In some embodiments suitable DNA vectors of the invention will contain asequence encoding the siRNA molecule of the invention and a stufferfragment. Suitable stuffer fragments of the invention include sequencesknown in the art including without limitation sequences which do notencode an expressed protein molecule; sequences which encode a normalcellular protein which would not have deleterious effect on the celltypes in which it was expressed; and sequences which would notthemselves encode a functional siRNA duplex molecule.

In one embodiment, suitable DNA molecules for use in AAV vectors will beless than about 5 kilobases (kb) in size and will include, for example,a stuffer sequence and a sequence encoding a siRNA molecule of theinvention. For example, in order to prevent any packaging of AAV genomicsequences containing the rep and cap genes, a plasmid containing the repand cap DNA fragment may be modified by the inclusion of a stufferfragment as is known in the art into the AAV genome which causes the DNAto exceed the length for optimal packaging. Thus, the helper fragment isnot packaged into AAV virions. This is a safety feature, ensuring thatonly a recombinant AAV vector genome that does not exceed optimalpackaging size is packaged into virions. An AAV helper fragment thatincorporates a stuffer sequence can exceed the wild-type genome lengthof 4.6 kb, and lengths above 105% of the wild-type will generally not bepackaged. The stuffer fragment can be derived from, for example, suchnon-viral sources as the Lac-Z or beta-galactosidase gene.

In one embodiment, the selected nucleotide sequence is operably linkedto control elements that direct the transcription or expression thereofin the subject in vivo. Such control elements can comprise controlsequences normally associated with the selected gene. Alternatively,heterologous control sequences can be employed. Useful heterologouscontrol sequences generally include those derived from sequencesencoding mammalian or viral genes. Examples include, but are not limitedto, the SV40 early promoter, mouse mammary tumor virus LTR promoter;adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, polII promoters, pol III promoters, synthetic promoters, hybrid promoters,and the like. In addition, sequences derived from nonviral genes, suchas the murine metallothionein gene, will also find use herein. Suchpromoter sequences are commercially available from, e.g., Stratagene(San Diego, Calif.).

In one embodiment, both heterologous promoters and other controlelements, such as CNS-specific and inducible promoters, enhancers andthe like, will be of particular use. Examples of heterologous promotersinclude the CMB promoter. Examples of CNS-specific promoters includethose isolated from the genes from myelin basic protein (MBP), glialfibrillary acid protein (GFAP), and neuron specific enolase (NSE).Examples of inducible promoters include DNA responsive elements forecdysone, tetracycline, hypoxia and aufin.

In one embodiment, the AAV expression vector which harbors the DNAmolecule of interest bounded by AAV ITRs, can be constructed by directlyinserting the selected sequence(s) into an AAV genome which has had themajor AAV open reading frames (“ORFs”) excised therefrom. Other portionsof the AAV genome can also be deleted, so long as a sufficient portionof the ITRs remain to allow for replication and packaging functions.Such constructs can be designed using techniques well known in the art.See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; InternationalPublication Nos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769(published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol.8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring HarborLaboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., supra. Forexample, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mMMgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 μMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian CNS cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al.Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook etal. (1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et. al. (1981) Gene 13:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

In one embodiment, suitable host cells for producing rAAV virionsinclude microorganisms, yeast cells, insect cells, and mammalian cells,that can be, or have been, used as recipients of a heterologous DNAmolecule. The term includes the progeny of the original cell which hasbeen transfected. Thus, a “host cell” as used herein generally refers toa cell which has been transfected with an exogenous DNA sequence. Cellsfrom the stable human cell line, 293 (readily available through, e.g.,the American Type Culture Collection under Accession Number ATCCCRL1573) can be used in the practice of the present invention.Particularly, the human cell line 293 is a human embryonic kidney cellline that has been transformed with adenovirus type-5 DNA fragments(Graham et al. (1977) J. Gen. Virol. 36:59), and expresses theadenoviral E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The293 cell line is readily transfected, and provides a particularlyconvenient platform in which to produce rAAV virions.

In one embodiment, host cells containing the above-described AAVexpression vectors are rendered capable of providing AAV helperfunctions in order to replicate and encapsidate the nucleotide sequencesflanked by the AAV ITRs to produce rAAV virions. AAV helper functionsare generally AAV-derived coding sequences which can be expressed toprovide AAV gene products that, in turn, function in trans forproductive AAV replication. AAV helper functions are used herein tocomplement necessary AAV functions that are missing from the AAVexpression vectors. Thus, AAV helper functions include one, or both ofthe major AAV ORFs, namely the rep and cap coding regions, or functionalhomologues thereof.

The Rep expression products have been shown to possess many functions,including, among others: recognition, binding and nicking of the AAVorigin of DNA replication; DNA helicase activity; and modulation oftranscription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

The term “AAV helper construct” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor lytic AAV replication; however, helper constructs lack AAV ITRs andcan neither replicate nor package themselves. AAV helper constructs canbe in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

In one embodiment, AAV helper functions are introduced into the hostcell by transfecting the host cell with an AAV helper construct eitherprior to, or concurrently with, the transfection of the AAV expressionvector. AAV helper constructs are thus used to provide at leasttransient expression of AAV rep and/or cap genes to complement missingAAV functions that are necessary for productive AAV infection. AAVhelper constructs lack AAV ITRs and can neither replicate nor packagethemselves. These constructs can be in the form of a plasmid, phage,transposon, cosmid, virus, or virion. A number of AAV helper constructshave been described, such as the commonly used plasmids pAAV/Ad andpIM29+45 which encode both Rep and Cap expression products. See, e.g.,Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991)J. Virol. 65:2936-2945. A number of other vectors have been describedwhich encode Rep and/or Cap expression products. See, e.g., U.S. Pat.No. 5,139,941.

In one embodiment, both AAV expression vectors and AAV helper constructscan be constructed to contain one or more optional selectable markers.Suitable markers include genes which confer antibiotic resistance orsensitivity to, impart color to, or change the antigenic characteristicsof those cells which have been transfected with a nucleic acid constructcontaining the selectable marker when the cells are grown in anappropriate selective medium. Several selectable marker genes that areuseful in the practice of the invention include the hygromycin Bresistance gene (encoding Aminoglycoside phosphotransferase (APH)) thatallows selection in mammalian cells by conferring resistance to G418(available from Sigma, St. Louis, Mo.). Other suitable markers are knownto those of skill in the art.

In one embodiment, the host cell (or packaging cell) is rendered capableof providing non AAV derived functions, or “accessory functions,” inorder to produce rAAV virions. Accessory functions are non AAV derivedviral and/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

In one embodiment, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

In one embodiment, accessory functions are provided using an accessoryfunction vector. Accessory function vectors include nucleotide sequencesthat provide one or more accessory functions. An accessory functionvector is capable of being introduced into a suitable host cell in orderto support efficient AAV virion production in the host cell. Accessoryfunction vectors can be in the form of a plasmid, phage, transposon orcosmid. Accessory vectors can also be in the form of one or morelinearized DNA or RNA fragments which, when associated with theappropriate control elements and enzymes, can be transcribed orexpressed in a host cell to provide accessory functions. See, forexample, International Publication No. WO 97/17548, published May 15,1997.

In one embodiment, nucleic acid sequences providing the accessoryfunctions can be obtained from natural sources, such as from the genomeof an adenovirus particle, or constructed using recombinant or syntheticmethods known in the art. In this regard, adenovirus-derived accessoryfunctions have been widely studied, and a number of adenovirus genesinvolved in accessory functions have been identified and partiallycharacterized. See, e.g., Carter, B. J. (1990) “Adeno-Associated VirusHelper Functions,” in CRC Handbook of Parvoviruses, vol. I (P. Tijssen,ed.), and Muzyczka, N. (1992) Curr. Topics. Microbiol and Immun.158:97-129. Specifically, early adenoviral gene regions E1 a, E2a, E4,VAI RNA and, possibly, E1b are thought to participate in the accessoryprocess. Janik et al. (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929.Herpesvirus-derived accessory functions have been described. See, e.g.,Young et al (1979) Prog. Med. Virol. 25:113. Vaccinia virus-derivedaccessory functions have also been described. See, e.g., Carter, B. J.(1990), supra., Schlehofer et al. (1986) Virology 152:110-117.

In one embodiment, as a consequence of the infection of the host cellwith a helper virus, or transfection of the host cell with an accessoryfunction vector, accessory functions are expressed which transactivatethe AAV helper construct to produce AAV Rep and/or Cap proteins. The Repexpression products excise the recombinant DNA (including the DNA ofinterest) from the AAV expression vector. The Rep proteins also serve toduplicate the AAV genome. The expressed Cap proteins assemble intocapsids, and the recombinant AAV genome is packaged into the capsids.Thus, productive AAV replication ensues, and the DNA is packaged intorAAV virions.

In one embodiment, following recombinant AAV replication, rAAV virionscan be purified from the host cell using a variety of conventionalpurification methods, such as CsCl gradients. Further, if infection isemployed to express the accessory functions, residual helper virus canbe inactivated, using known methods. For example, adenovirus can beinactivated by heating to temperatures of approximately 60.degrees C.for, e.g., 20 minutes or more. This treatment effectively inactivatesonly the helper virus since AAV is extremely heat stable while thehelper adenovirus is heat labile. The resulting rAAV virions are thenready for use for DNA delivery to the CNS (e.g., cranial cavity) of thesubject.

Methods of delivery of viral vectors include, but are not limited to,intra-arterial, intramuscular, intravenous, intranasal and oral routes.Generally, rAAV virions may be introduced into cells of the CNS usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with CNS cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest can be screened using conventional techniques such as Southernblots and/or PCR, or by using selectable markers. Transduced cells canthen be formulated into pharmaceutical compositions, described morefully below, and the composition introduced into the subject by varioustechniques, such as by grafting, intramuscular, intravenous,subcutaneous and intraperitoneal injection.

In one embodiment, for in vivo delivery, the rAAV virions are formulatedinto pharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle or by injection into the CNS.

In one embodiment, viral vectors of the invention are delivered to theCNS via convection-enhanced delivery (CED) systems that can efficientlydeliver viral vectors, e.g., AAV, over large regions of a subject'sbrain (e.g., striatum and/or cortex). As described in detail andexemplified below, these methods are suitable for a variety of viralvectors, for instance AAV vectors carrying therapeutic genes (e.g.,siRNAs).

Any convection-enhanced delivery device may be appropriate for deliveryof viral vectors. In one embodiment, the device is an osmotic pump or aninfusion pump. Both osmotic and infusion pumps are commerciallyavailable from a variety of suppliers, for example Alzet Corporation,Hamilton Corporation, Aiza, Inc., Palo Alto, Calif.). Typically, a viralvector is delivered via CED devices as follows. A catheter, cannula orother injection device is inserted into CNS tissue in the chosensubject. In view of the teachings herein, one of skill in the art couldreadily determine which general area of the CNS is an appropriatetarget. For example, when delivering AAV vector encoding a therapeuticgene to treat PD, the striatum is a suitable area of the brain totarget. Stereotactic maps and positioning devices are available, forexample from ASI Instruments, Warren, Mich. Positioning may also beconducted by using anatomical maps obtained by CT and/or MRI imaging ofthe subject's brain to help guide the injection device to the chosentarget. Moreover, because the methods described herein can be practicedsuch that relatively large areas of the brain take up the viral vectors,fewer infusion cannula are needed. Since surgical complications arerelated to the number of penetrations, the methods described herein alsoserve to reduce the side effects seen with conventional deliverytechniques.

In one embodiment, pharmaceutical compositions will comprise sufficientgenetic material to produce a therapeutically effective amount of thesiRNA of interest, i.e., an amount sufficient to reduce or amelioratesymptoms of the disease state in question or an amount sufficient toconfer the desired benefit. The pharmaceutical compositions will alsocontain a pharmaceutically acceptable excipient. Such excipients includeany pharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, sorbitol,Tween80, and liquids such as water, saline, glycerol and ethanol.Pharmaceutically acceptable salts can be included therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

As is apparent to those skilled in the art in view of the teachings ofthis specification, an effective amount of viral vector which must beadded can be empirically determined. Administration can be effected inone dose, continuously or intermittently throughout the course oftreatment. Methods of determining the most effective means and dosagesof administration are well known to those of skill in the art and willvary with the viral vector, the composition of the therapy, the targetcells, and the subject being treated. Single and multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

It should be understood that more than one transgene could be expressedby the delivered viral vector. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered tothe CNS as described herein. Furthermore, it is also intended that theviral vectors delivered by the methods of the present invention becombined with other suitable compositions and therapies.

BRIEF DESCRIPTION OF THE FIGURES

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. siRNA expressed from CMV promoter constructs and in vitroeffects. (A) A cartoon of the expression plasmid used for expression offunctional siRNA in cells. The CMV promoter was modified to allow closejuxtaposition of the hairpin to the transcription initiation site, and aminimal polyadenylation signal containing cassette was constructedimmediately 3′ of the MCS (mCMV, modified CMV; mpA, minipA). (B, C)Fluorescence photomicrographs of HEK293 cells 72 h after transfection ofpEGFPN1 and pCMVβ gal (control), or pEGFPN1 and pmCMVsiGFPmpA,respectively. (D) Northern blot evaluation of transcripts harvested frompmCMVsiGFPmpA (lanes 3, 4) and pmCMVsiβgalmpA (lane 2) transfectedHEK293 cells. Blots were probed with ³²P-labeled sense oligonucleotides.Antisense probes yielded similar results (not shown). Lane 1,³²P-labeled RNA markers. AdsiGFP infected cells also possessedappropriately sized transcripts (not shown). (E) Northern blot forevaluation of target mRNA reduction by siRNA (upper panel). The internalcontrol GAPDH is shown in the lower panel. HEK293 cells were transfectedwith pEGFPN1 and pmCMVsiGFPmpA, expressing siGFP, or plasmids expressingthe control siRNA as indicated. pCMVeGFPx, which expresses siGFPx,contains a large poly(A) cassette from SV40 large T and an unmodifiedCMV promoter, in contrast to pmCMVsiGFPmpA shown in (A). (F) Westernblot with anti-GFP antibodies of cell lysates harvested 72 h aftertransfection with pEGFPN1 and pCMVsiGFPmpA, or pEGFPN1 andpmCMVsiβglucmpA. (G, H) Fluorescence photomicrographs of HEK293 cells 72h after transfection of pEGFPN1 and pCMVsiGFPx, or pEGFPN1 andpmCMVsiβglucmpA, respectively. (I, J) siRNA reduces expression fromendogenous alleles. Recombinant adenoviruses were generated frompmCMVsiβglucmpA and pmCMVsiGFPmpA and purified. HeLa cells were infectedwith 25 infectious viruses/cell (MOI=25) or mock-infected (control) andcell lysates harvested 72 h later. (I) Northern blot for β-glucuronidasemRNA levels in Adsiβgluc and AdsiGFP transduced cells. GAPDH was used asan internal control for loading. (J) The concentration ofβ-glucuronidase activity in lysates quantified by a fluorometric assay.(Stein 1999).

FIG. 2. Viral vectors expressing siRNA reduce expression from transgenicand endogenous alleles in vivo. Recombinant adenovirus vectors wereprepared from the siGFP and siβgluc shuttle plasmids described inFIG. 1. (A) Fluorescence microscopy reveals diminution of eGFPexpression in vivo. In addition to the siRNA sequences in the E1 regionof adenovirus, RFP expression cassettes in E3 facilitate localization ofgene transfer. Representative photomicrographs of eGFP (left), RFP(middle), and merged images (right) of coronal sections from miceinjected with adenoviruses expressing siGFP (top panels) or siβgluc(bottom panels) demonstrate siRNA specificity in eGFP transgenic micestriata after direct brain injection. (B) Full coronal brain sections (1mm) harvested from AdsiGFP or Adsiβgluc injected mice were split intohemisections and both ipsilateral (i1) and contralateral (c1) portionsevaluated by western blot using antibodies to GFP. Actin was used as aninternal control for each sample. (C) Tail vein injection of recombinantadenoviruses expressing siβgluc directed against mouse β-glucuronidase(AdsiMuβgluc) reduces endogenous β-glucuronidase RNA as determined byNorthern blot in contrast to control-treated (Adsiβgal) mice.

FIG. 3. siGFP gene transfer reduces Q19-eGFP expression in cell lines.PC12 cells expressing the polyglutamine repeat Q19 fused to eGFP(eGFP-Q19) under tetracycline repression (A, bottom left) were washedand dox-free media added to allow eGFP-Q19 expression (A, top left).Adenoviruses were applied at the indicated multiplicity of infection(MOI) 3 days after dox removal. (A) eGFP fluorescence 3 days afteradenovirus-mediated gene transfer of Adsiβgluc (top panels) or AdsiGFP(bottom panels). (B, C) Western blot analysis of cell lysates harvested3 days after infection at the indicated MOIs demonstrate adose-dependent decrease in GFP-Q19 protein levels. NV, no virus. Toplanes, eGFP-Q19. Bottom lanes, actin loading controls. (D) Quantitationof eGFP fluorescence. Data represent mean total area fluorescence±standard deviation in 4 low power fields/well (3 wells/plate).

FIG. 4. siRNA mediated reduction of expanded polyglutamine proteinlevels and intracellular aggregates. PC12 cells expressingtet-repressible eGFP-Q80 fusion proteins were washed to removedoxycycline and adenovirus vectors expressing siRNA were applied 3 dayslater. (A-D) Representative punctate eGFP fluorescence of aggregates inmock-infected cells (A), or those infected with 100 MOI of Adsiβgluc(B), AdsiGFPx (C) or Adsiβgal (D). (E) Three days after infection ofdox-free eGFP-Q80 PC12 cells with AdsiGFP, aggregate size and number arenotably reduced. (F) Western blot analysis of eGFP-Q80 aggregates(arrowhead) and monomer (arrow) following Adsiβgluc or AdsiGFP infectionat the indicated MOIs demonstrates dose dependent siGFP-mediatedreduction of GFP-Q80 protein levels. (G) Quantification of the totalarea of fluorescent inclusions measured in 4 independent fields/well 3days after virus was applied at the indicated MOIs. The data aremean±standard deviation.

FIG. 5. (A) Allele-specific silencing of mutant huntingtin by siRNA.PC6-3 cells were co-transfected with plasmids expressing siRNA specificfor the polymorphism encoding the transcript for mutant huntingtin. (B)The original target for testing hairpins with putative specificity forthe 3 GAG-repeat disease linked polymorphism, shEx58.1 and shEx58.2. Inthis preliminary test, shEx58.1 is best.

FIG. 6. Silencing ataxin-1. (A) Cartoon of the ataxin-1 cDNA and regionstested for silencing (lines). The CAG repeat region is indicated. Themost effective hairpins identified, F10 and F11, are bolded. (B)Screening of shSCA1s for ataxin-1 silencing. HEK 293 cells weretransfected with sbRNA- and ataxin-1-expressing plasmids (4:1 ratio),and FLAG-tagged ataxin-1 (ataxin-1FLAG) expression was screened bywestern blot two days later. Actin was used as a loading control. ShLacZwas included as a negative hairpin control. Data shown are fromU6-expressed shRNAs. (C) Dose dependent decline in hSCA-1 mRNA asassessed by Q-RTPCR. HEK 293 cells were transfected with shRNA- andataxin-1-expressing plasmids at the ratios indicated, and RNA isolated24 hrs later. RNA levels were measured by Q-PCR as described in themethods. (D) Comparison of mCMV- and U6-expressed shRNAs in neuronalcells. PC6-3 cells were transfected with plasmids expressing theindicated shRNAs, and expression of ataxin assessed 2 days later bywestern blot. shCAG was targeted to the CAG repeat region and was usedas a positive control for silencing (E) The loop from miR23 improvessilencing from the hU6 promoter. HEK 293 cells were transfected withplasmids expressing the indicated hairpins and ataxin-1FLAG, andsilencing evaluated 2 days later by western blot. The loop improvessilencing of shSCA1.F10 and shSCA1.F11. (F) shSCA1.F10 and shSCA1.F11silence mutant (Q82) ataxin-1. HEK 293 cells were transfected withplasmids expressing the indicated hairpins, and a plasmid expressinghuman ataxin-1 with an expanded poly(Q) tract (FLAG-tagged). Silencingof the human mutant ataxin-1 was assessed by western blot 2 days later.

FIG. 7. AAV vectors for shRNA expression in vivo. (A) Cartoon of AAVconstruct. The construct for shSCA.F11mi and shLacZ expression wassimilar except that shSCA1.F10 ml was replaced with shSCA.F11mi orshLacZ sequences, respectively. Note that the hrGFP expression cassetteis distinct from the shRNA expression cassette. (B) AAVshSCA1 with hrGFPreporter leads to extensive transduction of cerebellar Purkinje cells(Purkinje cell layer denoted by arrowheads). Wildtype mice were injectedwith AAVshSCA1.F10 ml (left panel) or injected with saline (right panel)and sacrificed 3 weeks later to evaluate eGFP expression. g, granulecell layer; m, molecular layer. Bar=100 μm. (C) shSCA1 and shLacZtranscripts are expressed in vivo. Wildtype mice were injected withAAVshLacZ or AAVshSCA1.F10 ml, and RNA isolated from cerebella 10 dayslater. Northern blots were probed with 32P-labeled oligonucleotidesspecific for the antisense strand of the hairpin. L, RNA ladder; (sizesindicated at left). Lanes, 2 and 3, RNA from AAVshSCA1.F10 ml andAAVshLacZ transduced brains, respectively. The arrowhead denotes theunprocessed transcript, the arrow the processed siRNA. (D) Rotarodperformance of wildtype (triangles) and SCA1 (squares) mice treated withshRNA-expressing AAV1s or mock infected, as indicated in the legend.Mice were injected with virus or saline at age 7 weeks and re-testedevery two weeks (weeks 5, 11, 15, and 21 are shown). From weeks 11-21significant differences in performance between AAVshSCA1 and AAVshLacZtreated SCA1 mice were noted (P<0.001). There were no significantdifferences between wildtype mice treated with shLacZ (not shown),shSCA1.F10 ml or saline. For week 5, n=10 and 11 for shSCA1 and shLacZtreated SCA1 mice, respectively; n=6 and 5 for shSCA1 and controltreated age-matched wildtype littermates, respectively. For weeks 7-21,n=14 and 12 for shSCA1 and shLacZ treated SCA1 mice, respectively; n=12and 11 for shSCA1 and control treated age-matched wildtype littermates,respectively; n=9 for saline injected SCA1 mice. WT mice given shLacZwere not significantly different than WT mice treated with saline,shSCA1, or left untreated (data not shown).

FIG. 8. SCA1 neuropathology is improved by shRNAs directed to ataxin-1.(A) SCA and wildtype mice were injected with AAVshSCA1.F10 ml orAAVshLacZ at week 7, and sacrificed 9 weeks later for cerebellarpathology. Calbindin immunofluorescence (IF) (middle panels) and hrGFPexpression (top panels) were evaluated. Merged images (bottom panels)demonstrate that hrGFP+ molecular layers from AAVshSCA-injected SCA1mice have calbindin staining similar to wildtype mice. Panels arerepresentative of 100 or 40 sections evaluated for AAVshSCA1.F10ml-treated SCA1 or wildtype mice, respectively, and 80 sections fromAAVshLacZ-treated mice. Bar in upper left panel=50 μm and isrepresentative of all images. (B) The molecular layer width intransduced (solid bars), and untransduced (open bars) lobules fromwildtype and SCA1 mice was measured. The data demonstrate significantprotection following shSCA1.F10 ml therapy. **, P<0.001. Numbers belowbars refer to numbers of sections measured/group. Molecular layer widthsfrom wildtype mice given AAVs expressing shLacZ or shSCA1.F10 ml wereindistinguishable and were pooled for comparison to SCA1 mice cerebella(designated shRNA). (C) Photomicrographs shown in A, and FIG. 10, arefrom the region boxed.

FIG. 9. Effects of shSCA1.F10 ml and shSCA1.F11mi on ataxin-1 expressionin mice cerebella. SCA1 transgenic or wildtype mice were injected withthe indicated shRNA-expressing AAVs, and cerebella harvested 1 weeklater and processed for hrGFP fluorescence, and ataxin-1 IF. The toppanels are from untreated SCA1 mice. The arrowheads in the middle andmerged panels depict pairs of Purkinje cells, one transduced (hrGFP+),and one untransduced (hrGFP−), highlighting the extent of reduction intransgenic ataxin-1 (Q82) expression from mice injected withAAVshSCA1.F10 ml and AAVshSCA1.F11mi, but not AAVshLacZ. Mouse ataxin-1IF is weak, but notable, in wildtype mice (lower middle panel), and itsexpression is not reduced following shSCA1.F11mi-treatment. Bar=25 μmand refers to all panels.

FIG. 10. RNAi reduces intranuclear inclusions in transduced cells. (A)Inclusions in transduced (hrGFP+) vs. untransduced cells. Brains fromSCA1 and wildtype mice were harvested 9 weeks after gene transfer (16weeks of age) and processed to evaluate hrGFP fluorescence and ataxin-1IF. Bar=25 μm and is representative of all images. (B) Highermagnification of merged hrGFP and ataxin-1 positive cells. There arepunctate ataxin-1 inclusions and robust nuclear staining in untransduced(Un) or AAVshLacZ transduced SCA1 Purkinje cells (top and bottom,respectively), but not AAVshSCA1.F10 ml transduced ones (middle panel;see also FIG. 11). Numbers in lower left refer to % intranuclearinclusion-positive Purkinje cells in ˜400 cells scored.

FIG. 11. Reductions in ataxin-1 inclusions in SCA1 mice requirestransduction. Sections from SCA1 mice injected 9 weeks earlier withAAVshSCA1.F10 ml were evaluated for hrGFP expression to identifytransduced cells, and ataxin-1 inclusions using IF, as described in theMethods and to the legend of FIG. 4. The photomicrographs demonstratethat ataxin-1 inclusions are noted in untransduced cells, but nottransduced cells, from AAVshSCA1.F10 ml-treated mice Bar=25 μm.

FIG. 12. PCR method for cloning hairpins. A 79 nt primer is used withthe Ampr template. Pfu and DMSO are used in the amplification reaction.Products are ligated directly into pCR-Blunt Topo (Invitrogen) and Kanrresistant colonies picked and sequenced. Positive clones can be useddirectly.

FIG. 13. Reduction of eGFP inclusions after transduction with 25, 50 or100 viruses/cell into cultures with pre-formed aggregates. Notedose-dependent response with shGFP vectors only.

FIG. 14. Regulated RNAi. Two Teto2 sequences were placed up- anddown-stream of the TATA box of the H1 promoter element (cartoon). Eithercontrol shRNA or shGFP was placed into the cassette for expression ofhairpins. Plasmids expressing GFP and the hairpin constructs weretransfected into a cell line expressing the TetR (tet-repressor). GFPfluorescence (left panels) or western blot (right panels) was evaluatedin the absence (TetR binding) or presence (TetR off) of doxycycline.

FIG. 15. Top, FIV construct. Bottom, AAV construct. Both express thehrGFP reporter so that transduced cells can be readily evaluated forshRNA efficacy (as in FIGS. 3 and 4).

FIG. 16. RNAi-mediated suppression of expanded CAG repeat containinggenes. Expanded CAG repeats are not direct targets for preferentialinactivation (A), but a linked SNP can be exploited to generate siRNAthat selectively silences mutant ataxin-3 expression (B-F). (A)Schematic of cDNA encoding generalized polyQ-fluorescent proteinfusions. Bars indicate regions targeted by siRNAs. HeLa cellsco-transfected with Q80-GFP, Q19-RFP and the indicated siRNA. Nuclei arevisualized by DAPI staining (blue) in merged images. (B) Schematic ofhuman ataxin-3 cDNA with bars indicating regions targeted by siRNAs. Thetargeted SNP (G987C) is shown in color. In the displayed siRNAs, red orblue bars denote C or G respectively. In this Figure,AGCAGCAGCAGGGGGACCTATCAGGAC is SEQ ID NO:7, andCAGCAGCAGCAGCGGGACCTATCAGGAC is SEQ ID NO:8. (C) Quantitation offluorescence in Cos-7 cells transfected with wild type or mutantataxin-3-GFP expression plasmids and the indicated siRNA. Fluorescencefrom cells co-transfected with siMiss was set at one. Bars depict meantotal fluorescence from three independent experiments +/−standard errorof the mean (SEM). (D) Western blot analysis of cells co-transfectedwith the indicated ataxin-3 expression plasmids (top) and siRNAs(bottom). Appearance of aggregated, mutant ataxin-3 in the stacking gel(seen with siMiss and siG10) is prevented by siRNA inhibition of themutant allele. (E) Allele specificity is retained in the simulatedheterozygous state. Western blot analysis of Cos-7 cells cotransfectedwith wild-type (atx-3-Q28-GFP) and mutant (atx-Q166) expression plasmidsalong with the indicated siRNAs. (Mutant ataxin-3 detected with 1C2, anantibody specific for expanded polyQ, and wild-type ataxin-3 detectedwith anti-ataxin-3 antibody.) (F) Western blot of Cos-7 cellstransfected with Atx-3-GFP expression plasmids and plasmids encoding theindicated shRNA. The negative control plasmid, phU6-LacZi, encodes siRNAspecific for LacZ. Both normal and mutant protein were detected withanti-ataxin-3 antibody. Tubulin immunostaining shown as a loadingcontrol in panels (D)-(F).

FIG. 17. Primer sequences for in vitro synthesis of siRNAs using T7polymerase. All primers contain the following T7 promoter sequence attheir 3′ ends: 5′-TATAGTGAGTCGTATTA-3′ (SEQ ID NO:9). The followingprimer was annealed to all oligos to synthesize siRNAs:5′-TAATACGACTCACTATAG-3′ (SEQ ID NO:10).

FIG. 18. Inclusion of either two (siC7/8) or three (siC10) CAG tripletsat the 5′ end of ataxin-3 siRNA does not inhibit expression of unrelatedCAG repeat containing genes. (A) Western blot analysis of Cos-7 cellstransfected with CAG repeat-GFP fusion proteins and the indicated siRNA.Immunostaining with monoclonal anti-GFP antibody (MBL) at 1:1000dilution. (B) Western blot analysis of Cos-7 cells transfected withFlag-tagged ataxin-1-Q30, which is unrelated to ataxin-3, and theindicated siRNA. Immunostaining with anti-Flag monoclonal antibody(Sigma St. Louis, Mo.) at 1:1000 dilution. In panels (A) and (B),lysates were collected 24 hours after transfection. Tubulinimmunostaining shown as a loading control.

FIG. 19. shRNA-expressing adenovirus mediates allele-specific silencingin transiently transfected Cos-7 cells simulating the heterozygousstate. (A) Representative images of cells cotransfected to express wildtype and mutant ataxin-3 and infected with the indicated adenovirus at50 multiplicities of infection (MOI). Atx-3-Q28-GFP (green) is directlyvisualized and Atx-3-Q166 (red) is detected by immunofluorescence with1C2 antibody. Nuclei visualized with DAPI stain in merged images. Anaverage of 73.1% of cells co-expressed both ataxin-3 proteins withsiMiss. (B) Quantitation of mean fluorescence from 2 independentexperiments performed as in (A). (C) Western blot analysis ofviral-mediated silencing in Cos-7 cells expressing wild type and mutantataxin-3 as in (A). Mutant ataxin-3 detected with 1C2 antibody andwild-type human and endogenous primate ataxin-3 detected withanti-ataxin-3 antibody. (D) shRNA-expressing adenovirus mediatesallele-specific silencing in stably transfected neural cell lines.Differentiated PC12 neural cells expressing wild type (left) or mutant(right) ataxin-3 were infected with adenovirus (100 MOI) engineered toexpress the indicated hairpin siRNA. Shown are Western blotsimmunostained for ataxin-3 and GAPDH as loading control.

FIG. 20. Allele-specific siRNA suppression of a missense Tau mutation.(A) Schematic of human tau cDNA with bars indicating regions andmutations tested for siRNA suppression. Of these, the V337M regionshowed effective suppression and was further studied. Vertical barsrepresent microtubule binding repeat elements in Tau. In the displayedsiRNAs, blue and red bars denote A and C respectively. In this Figure,GTGGCCAGATGGAAGTAAAATC is SEQ ID NO:35, and GTGGCCAGGTGGAAGTAAAATC isSEQ ID NO:41. (B) Western blot analysis of cells co-transfected with WTor V337M Tau-EGFP fusion proteins and the indicated siRNAs. Cells werelysed 24 hr after transfection and probed with anti-tau antibody.Tubulin immunostaining is shown as loading control. (C) Quantitation offluorescence in Cos-7 cells transfected with wild type tau-EGFP ormutant V337M tau-EGFP expression plasmids and the indicated siRNAs. Barsdepict mean fluorescence and SEM from three independent experiments.Fluorescence from cells co-transfected with siMiss was set at one.

FIG. 21. Allele-specific silencing of Tau in cells simulating theheterozygous state. (A) Representative fluorescent images of fixed Helacells co-transfected with flag-tagged WT-Tau (red), V337M-Tau-GFP(green), and the indicated siRNAs. An average of 73.7% of cellsco-expressed both Tau proteins with siMiss. While siA9 suppresses bothalleles, siA9/C12 selectively decreased expression of mutant Tau only.Nuclei visualized with DAPI stain in merged images. (B) Quantitation ofmean fluorescence from 2 independent experiments performed as in (A).(C) Western blot analysis of cells co-transfected with Flag-WT-Tau andV337M-Tau-EGFP fusion proteins and the indicated siRNAs. Cells werelysed 24 hr after transfection and probed with anti-tau antibody.V337M-GFP Tau was differentiated based on reduced electrophoreticmobility due to the addition of GFP. Tubulin immunostaining is shown asa loading control.

FIG. 22. Schematic diagram of allele-specific silencing of mutantTorsinA by small interfering RNA (siRNA). In the disease state, wildtype and mutant alleles of TOR1A are both transcribed into mRNA. siRNAwith sequence identical to the mutant allele (deleted of GAG) shouldbind mutant mRNA selectively and mediate its degradation by theRNA-induced silencing complex (RISC) (circle). Wild type mRNA, notrecognized by the mutant-specific siRNA, will remain and continue to betranslated into normal TorsinA. The two adjacent GAG's in wild typeTOR1A alleles are shown as two parallelograms, one of which is deletedin mutant TOR1A alleles.

FIG. 23. Design and targeted sequences of siRNAs. Shown are the relativepositions and targeted mRNA sequences for each primer used in thisstudy. Mis-siRNA (negative control) does not target TA; com-siRNAtargets a sequence present in wild type and mutant TA; wt-siRNA targetsonly wild type TA; and three mutant-specific siRNAs (Mut A, B, C).preferentially target mutant TA. The pair of GAG codons near thec-terminus of wild type mRNA are shown in underlined gray and black,with one codon deleted in mutant mRNA.

FIG. 24. siRNA silencing of TAwt and TAmut in Cos-7 cells. (A) Westernblot results showing the effect of different siRNAs on GFP-TAwtexpression levels. Robust suppression is achieved with wt-siRNA andcom-siRNA, while the mutant-specific siRNAs MutA, (B) and (C) havemodest or no effect on GFP-TAwt expression. Tubulin loading controls arealso shown. (B) Similar experiments with cells expressing HA-TAmut,showing significant suppression by mutant-specific siRNAs and com-siRNAbut no suppression by the wild type-specific siRNA, wt-siRNA. (C)Quantification of results from at least three separate experiments as inA and B. (D) Cos-7 cells transfected with GFP-TAwt or GFP-TAmut anddifferent siRNAs visualized under fluorescence microscopy (200×).Representative fields are shown indicating allele-specific suppression.(E) Quantification of fluorescence signal from two different experimentsas in D.

FIG. 25. Allele-specific silencing by siRNA in the simulatedheterozygous state. Cos-7 cells were cotransfected with plasmidsencoding differentially tagged TAwt and TAmut, together with theindicated siRNA. (A) Western blot results analysis showing selectivesuppression of the targeted allele by wt-siRNA or mutC-siRNA. (B)Quantification of results from three experiments as in (A).

FIG. 26. RNAi reduces human huntingtin expression in vitro. (A) RNAsequence of shHD2.1. The 21 nucleotide antisense strand is cognate tonucleotides 416-436 of human htt mRNA (Genbank #NM 00211). (B and C)Northern and western blots demonstrate shHD2.1 mediated reduction ofHD-N171-82Q mRNA and protein expression, 48 h post-transfection oftarget- and shRNA-expressing plasmids. GAPDH and actin serve as loadingcontrols. (D) Western blots show that shHD2.1 inhibits expression offull-length human huntingtin protein, 48 h post-transfection. (E)ShHD2.1 induces dose-dependent reduction of human htt mRNA. Cells weretransfected with shLacZ- or shHD2.1-expressing plasmids in the indicatedamounts. Relative htt expression was determined by quantitative PCR 24 hlater. SEQ ID NO:56 is 5′-AAGAAAGAACUUUCAGCUACC-3′. SEQ ID NO:57 is5′-GGUAGCUGAAAGUUUCUU-3′. SEQ ID NO:58 is 5′-GAAGCUUG-3′. SEQ ID NO:59is 5′-AAGAAAGAACUUUCAGCUACCGAAGCUUGGGUAGCUGAAAGUUCU UUCUUUUUUUU-3′.

FIG. 27. AAV.shHD2.1 delivers widespread RNAi expression to mousestriatum. (A) AAV.shHD2.1 viral vector. ITR, inverted terminal repeat.(B) Northern blot showing shHD2.1 transcripts are expressed in vivo.Processed antisense (lower band) and unprocessed (upper band) shHD2.1transcripts in three different AAV.shHD2.1-injected mice. L, ladder; +,positive control oligo. Blot was probed with radiolabeled sense probe.(C) Typical AAV1 transduction pattern (hrGFP) in mouse brain. CC, corpuscallosum; LV, lateral ventricle.

FIG. 28. AAV.shHD2.1 eliminates accumulation of huntingtin-reactiveneuronal inclusions and reduces HD-N171-82Q mRNA in vivo. (A)Representative photomicrographs show htt-reactive inclusions (arrows) inHD striatal cells transduced with AAV.shLacZ-, but not AAV.shHD2.1.Scale bar, 20 μm. (B) Higher magnification photomicrograph from a(bottom, right) showing lack of htt-reactive inclusions in cellstransduced by AAV.shHD2.1. * serves as a marker for orientation. Scalebar, 20 μm. (C) Representative western blot demonstrates decreasedHD-N171-82Q expression in mouse striata transduced with AAV.shHD2.1compared to uninjected or AAV.shLacZ-injected striata. Prion protein wasused as a loading control to normalize for tissues expressing theHD-N171-82Q transgene. (D) AAV.shHD2.1-treated HD mice showed a 55%average reduction in HD-N171-82Q mRNA compared to AAV.shLacZ oruninjected HD mice. Data are means ±S.E.M. relative to uninjected HDsamples. *, difference from AAV.shHF2.1 samples, p<0.05 (ANOVA). (E)Mice were injected directly into cerebellum with AAV.shHD2.1 orAAV.shLacZ. Cerebellar sections confirm that AAV.shHD2.1, but notAAV.shLacZ, reduces htt immunoreactivity. GCL, granule cell layer; ML,molecular layer. Scale bar, 100 μm.

FIG. 29. AAV.shHD2.1 improves behavioral deficits in HD-N171-82Q mice.(A) Box plot. Bilateral striatal delivery of AAV.shHD2.1 improves stridelength in HD-N171-82Q mice. HD mice had significantly shorter stridelengths compared to WT. AAV.shHD2.1 mediated significant gaitimprovement relative to control-treated HD mice. *, p<0.0001 (ANOVA,Scheffe post-hoc). (B) Bilateral striatal delivery of AAV.shHD2.1significantly improves rotarod performance in HD-N171-82Q mice. OnlyAAV.shLacZ-injected and uninjected HD-N171-82Q declined significantlywith time. Data are means ±S.E.M.

FIG. 30. DNA sequences of huntingtin hairpins. The bases that areunderlined indicate changes from the native huntingtin sequence.

FIG. 31. PCR method for cloning hairpins. A 79 nt primer is used withthe Ampr template. Pfu and DMSO are used in the amplification reaction.Products are ligated directly into pCR-Blunt Topo (Invitrogen) and Kanrresistant colonies picked and sequenced. Positive clones can be useddirectly.

FIG. 32. Reduction of eGFP inclusions after transduction with 25, 50 or100 viruses/cell into cultures with pre-formed aggregates. Notedose-dependent response with shGFP vectors only.

FIG. 33. Regulated RNAi. Two Teto2 sequences were placed up- anddown-stream of the TATA box of the H1 promoter element (cartoon). Eithercontrol shRNA or shGFP was placed into the cassette for expression ofhairpins. Plasmids expressing GFP and the hairpin constructs weretransfected into a cell line expressing the TetR (tet-repressor). GFPfluorescence (left panels) or western blot (right panels) was evaluatedin the absence (TetR binding) or presence (TetR off) of doxycycline.

FIG. 34. Top, FIV construct. Bottom, AAV construct. Both express thehrGFP reporter so that transduced cells can be readily evaluated forshRNA efficacy (as in FIGS. 3 and 4).

FIG. 35. siRNA molecules specific for regions of the HD gene.

DETAILED DESCRIPTION OF THE INVENTION

Modulation of gene expression by endogenous, noncoding RNAs isincreasingly appreciated as a mechanism playing a role in eukaryoticdevelopment, maintenance of chromatin structure and genomic integrity(McManus, 2002). Recently, techniques have been developed to trigger RNAinterference (RNAi) against specific targets in mammalian cells byintroducing exogenously produced or intracellularly expressed siRNAs(Elbashir, 2001a, 2001b, 2001c; Brummelkamp, 2002). These methods haveproven to be quick, inexpensive and effective for knockdown experimentsin vitro and in vivo (Elbashir, 2001a, 2001b, 2001c; Brummelkamp, 2002;McCaffrey, 2002; Xia, 2002). The ability to accomplish selective genesilencing has led to the hypothesis that siRNAs might be employed tosuppress gene expression for therapeutic benefit (Xia, 2002; Jacque,2002; Gitlin, 2002).

RNA interference is now established as an important biological strategyfor gene silencing, but its application to mammalian cells has beenlimited by nonspecific inhibitory effects of long double-stranded RNA ontranslation. Moreover, delivery of interfering RNA has largely beenlimited to administration of RNA molecules. Hence, such administrationmust be performed repeatedly to have any sustained effect. The presentinventors have developed a delivery mechanism that results in specificsilencing of targeted genes through expression of small interfering RNA(siRNA). The inventors have markedly diminished expression of exogenousand endogenous genes in vitro and in vivo in brain and liver, andfurther apply this novel strategy to a model system of a major class ofneurodegenerative disorders, the polyglutamine diseases, to show reducedpolyglutamine aggregation in cells. This strategy is generally useful inreducing expression of target genes in order to model biologicalprocesses or to provide therapy for dominant human diseases.

Disclosed herein is a strategy that results in substantial silencing oftargeted alleles via siRNA. Use of this strategy results in markedlydiminished in vitro and in vivo expression of targeted alleles. Thisstrategy is useful in reducing expression of targeted alleles in orderto model biological processes or to provide therapy for human diseases.For example, this strategy can be applied to a major class ofneurodegenerative disorders, the polyglutamine diseases, as isdemonstrated by the reduction of polyglutamine aggregation in cellsfollowing application of the strategy. As used herein the term“substantial silencing” means that the mRNA of the targeted allele isinhibited and/or degraded by the presence of the introduced siRNA, suchthat expression of the targeted allele is reduced by about 10% to 100%as compared to the level of expression seen when the siRNA is notpresent. Generally, when an allele is substantially silenced, it willhave at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%,e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 100% reduction expression as compared to when the siRNA isnot present. As used herein the term “substantially normal activity”means the level of expression of an allele when an siRNA has not beenintroduced to a cell.

Dominantly inherited diseases, including polyQ neurodegenerativedisorders, are ideal candidates for siRNA-based therapy. The polyQneurodegenerative disorders include at least nine inherited disorderscaused by CAG repeat expansions that encode polyQ in the diseaseprotein. PolyQ expansion confers a dominant toxic property on the mutantprotein that is associated with aberrant accumulation of the diseaseprotein in neurons (Zoghbi, 2000). All polyQ diseases are progressive,ultimately fatal disorders that typically begin in adulthood. Huntingtondisease (HD) is the best known polyQ disease, but at least sevenhereditary ataxias and one motor neuron disease are also due to CAGrepeat/polyQ expansion. Although the clinical features and patterns ofneuronal degeneration differ among the diseases, increasing evidencesuggests that polyQ diseases share important pathogenic features. Inparticular, expansion of the CAG repeat/polyQ domain confers upon theencoded protein a dominant toxic property. Thus as a therapeuticstrategy, efforts to lower expression of the mutant gene product priorto cell death could be highly beneficial to patients.

Dominantly inherited diseases are ideal candidates for siRNA-basedtherapy. To explore the utility of siRNA in inherited human disorders,the present inventors employed cellular models to test whether mutantalleles responsible for these dominantly-inherited human disorders couldbe specifically targeted. First, three classes of dominantly inherited,untreatable neurodegenerative diseases were examined: polyglutamine(polyQ) neurodegeneration in MJD/SCA3, Huntington's disease andfrontotemporal dementia with parkinsonism linked to chromosome 17(FTDP-17). Machado-Joseph disease is also known as SpinocerebellarAtaxia Type 3 (The HUGO official name is MJD). The gene involved isMJD1, which encodes for the protein ataxin-3 (also called Mjd1p).Huntington's disease is due to expansion of the CAG repeat motif in exon1 of huntingtin. In 38% of patients a polymorphism exists in exon 58 ofthe huntingtin gene, allowing for allele specific targeting.Frontotemporal dementia (sometimes with parkinsonism, and linked tochromosome 17, so sometimes called FTDP-17) is due to mutations in theMAPT1 gene that encodes the protein tau.

The polyQ neurodegenerative disorders include at least nine diseasescaused by CAG repeat expansions that encode polyQ in the diseaseprotein. PolyQ expansion confers a dominant toxic property on the mutantprotein that is associated with aberrant accumulation of the diseaseprotein in neurons (Zoghbi, 2000). In FTDP-17, Tau mutations lead to theformation of neurofibrillary tangles accompanied by neuronal dysfunctionand degeneration (Poorkaj, 1998; Hutton, 1998). The precise mechanismsby which these mutant proteins cause neuronal injury are unknown, butconsiderable evidence suggests that the abnormal proteins themselvesinitiate the pathogenic process (Zoghbi, 2000). Accordingly, eliminatingexpression of the mutant protein by siRNA or other means slows orprevents disease (Yamamoto, 2000). However, because many dominantdisease genes also encode essential proteins (e.g. Nasir, 1995)siRNA-mediated approaches were developed that selectively inactivatemutant alleles, while allowing continued expression of the wild typeproteins ataxin-3 and huntingtin.

Second, the dominantly-inherited disorder DYT1 dystonia was studied.DYT1 dystonia is also known as Torsion dystonia type 1, and is caused bya GAG deletion in the TOR1A gene encoding torsinA. DYT1 dystonia is themost common cause of primary generalized dystonia. DYT1 usually presentsin childhood as focal dystonia that progresses to severe generalizeddisease (Fahn, 1998; Klein, 2002a). With one possible exception (Leung,2001; Doheny, 2002; Klein, 2002), all cases of DYT1 result from a commonGAG deletion in TOR1A, eliminating one of two adjacent glutamic acidsnear the C-terminus of the protein TorsinA (TA) (Ozelius, 1997).Although the precise cellular function of TA is unknown, it seems clearthat mutant TA (TAmut) acts through a dominant-negative ordominant-toxic mechanism (Breakefield, 2001).

Several characteristics of DYT1 make it an ideal disease in which to usesiRNA-mediated gene silencing as therapy. Of greatest importance, thedominant nature of the disease suggests that a reduction in mutant TA,whatever the precise pathogenic mechanism proves to be, is helpful.Moreover, the existence of a single common mutation that deletes a fullthree nucleotides suggested it might be feasible to design siRNA thatspecifically targets the mutant allele and is applicable to all affectedpersons. Finally, there is no effective therapy for DYT1, a relentlessand disabling disease.

As outlined in the strategy in FIG. 11, the inventors developed siRNAthat would specifically eliminate production of protein from the mutantallele. By exploiting the three base pair difference between wild typeand mutant alleles, the inventors successfully silenced expression ofthe mutant protein (TAmut) without interfering with expression of thewild type protein (TAwt). Because TAwt may be an essential protein it iscritically important that efforts be made to silence only the mutantallele. This allele-specific strategy has obvious therapeutic potentialfor DYT1 and represents a novel and powerful research tool with which toinvestigate the function of TA and its dysfunction in the disease state.

Expansions of poly-glutamine tracts in proteins that are expressed inthe central nervous system can cause neurodegenerative diseases. Someneurodegenerative diseases are caused by a (CAG), repeat that encodespoly-glutamine in a protein include Huntington disease (HD),spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7), spinal and bulbarmuscular atrophy (SBMA), and dentatorubropallidoluysian atrophy (DRPLA).In these diseases, the poly-glutamine expansion in a protein confers anovel toxic property upon the protein. Studies indicate that the toxicproperty is a tendency for the disease protein to misfold and formaggregates within neurons.

CAG triplet repeat expansion in exon 1 of Hdh causes Huntington'sdisease. Clinical characteristics of HD include progressive loss ofstriatal neurons and later, cortical thinning. Adult patients showchoreiform movements, impaired coordination, progressive dementia andother psychiatric disturbances. The symptoms of juvenile HD patientsinclude bradykinesia, dystonia and seizures. HD is a uniformly fataldisease, with death occurring one to two decades after disease onset.

The Hdh locus is on chromosome 4, spans 180 kb over 67 exons and encodesthe protein huntingtin (htt). In non-HD individuals, the CAG repeatregion is less than 35 CAG repeats. Expansions of 36 to ˜50 repeats, orgreater than ˜50, cause late or early onset disease, respectively. Theinverse correlation of repeat length with age of disease onset is acommon characteristic of the CAG repeat disorders, and one that isrecapitulated in mouse models. Evidence indicates that HD also may be adose-dependent process. For example, in transgenic mouse models of polyQdisease, phenotypic severity usually correlates with expression levelsof the disease protein, and homozygous transgenic mice develop diseasemore rapidly than heterozygous mice. In addition, the very rare humancases of homozygosity for polyQ disease suggest that disease severitycorrelates with the level of disease protein expression, againsupporting the notion that reducing mutant protein expression would beclinically beneficial.

The function of htt is not known. It is clear from mouse models,however, that it is required during gastrulation, neurogenesis and inpostnatal brain. Htt knock-out mice die during development. Also,removal of htt via Cre recombinase-mediated excision of a floxed Hdhallele causes progressive postnatal neurodegeneration. A CAG expansionintroduced into the mouse allele (a knock-in) does not impairneurogenesis unless wildtype htt expression is reduced from normallevels, suggesting that the expanded allele does not impair wildtype httfunction in neurogenesis. In adult mice mutant htt causes progressivedepletion of normal htt. Htt is important in vesicle trafficking, NMDAreceptor modulation, and regulation of BDNF transcription, and theexpression of many genes is affected in the CNS of HD mice.

The therapeutic promise of silencing the mutant gene (and its toxicproperty) is best demonstrated in a tetracycline-regulated mouse modelof HD (Yamamoto 2000). When mutant htt is inducibly expressed in thesemice, pathological and behavioral features of the disease develop overtime, including the characteristic formation of neuronal inclusions andabnormal motor behavior (Yamamoto 2000, Orr 2000). However, whenexpression of the transgene is repressed in affected mice, thepathological and behavioral features of disease fully resolve (Yamamoto2000). This result indicates that if expression of mutant polyQ proteincan be halted, protein clearance mechanisms within neurons can eliminatethe aggregated mutant protein, and possibly normalize mutant htt-inducedchanges. It also suggests that gene silencing approaches may bebeneficial even for individuals with fairly advanced disease.

One of skill in the art can select additional target sites forgenerating siRNA specific for other alleles beyond those specificallydescribed in the experimental examples. Such allele-specific siRNAs madebe designed using the guidelines provided by Ambion (Austin, Tex.).Briefly, the target cDNA sequence is scanned for target sequences thathad AA di-nucleotides. Sense and anti-sense oligonucleotides aregenerated to these targets (AA+3′ adjacent 19 nucleotides) thatcontained a G/C content of 35 to 55%. These sequences are then comparedto others in the human genome database to minimize homology to otherknown coding sequences (BLAST search).

To accomplish intracellular expression of the therapeutic siRNA, an RNAmolecule is constructed containing two complementary strands or ahairpin sequence (such as a 21-bp hairpin) representing sequencesdirected against the gene of interest. The siRNA, or a nucleic acidencoding the siRNA, is introduced to the target cell, such as a diseasedbrain cell. The siRNA reduces target mRNA and protein expression.

The construct encoding the therapeutic siRNA is configured such that theone or more strands of the siRNA are encoded by a nucleic acid that isimmediately contiguous to a promoter. In one example, the promoter is apol II promoter. If a pol II promoter is used in a particular construct,it is selected from readily available pol II promoters known in the art,depending on whether regulatable, inducible, tissue or cell-specificexpression of the siRNA is desired. The construct is introduced into thetarget cell, allowing for diminished target-gene expression in the cell.

It was surprising that a pol II promoter would be effective. While smallRNAs with extensive secondary structure are routinely made from Pol IIIpromoters, there is no a priori reason to assume that small interferingRNAs could be expressed from pol II promoters. Pol III promotersterminate in a short stretch of Ts (5 or 6), leaving a very small 3′ endand allowing stabilization of secondary structure. Polymerase IItranscription extends well past the coding and polyadenylation regions,after which the transcript is cleaved. Two adenylation steps occur,leaving a transcript with a tail of up to 200 As. This string of Aswould of course completely destabilize any small, 21 base pair hairpin.Therefore, in addition to modifying the promoter to minimize sequencesbetween the transcription start site and the siRNA sequence (therebystabilizing the hairpin), the inventors also extensively modified thepolyadenylation sequence to test if a very short polyadenylation couldoccur. The results, which were not predicted from prior literature,showed that it could.

The present invention provides an expression cassette containing anisolated nucleic acid sequence encoding a small interfering RNA molecule(siRNA) targeted against a gene of interest. The siRNA may form ahairpin structure that contains a duplex structure and a loop structure.The loop structure may contain from 4 to 10 nucleotides, such as 4, 5 or6 nucleotides. The duplex is less than 30 nucleotides in length, such asfrom 19 to 25 nucleotides. The siRNA may further contain an overhangregion. Such an overhang may be a 3′ overhang region or a 5′ overhangregion. The overhang region may be, for example, from 1 to 6 nucleotidesin length. The expression cassette may further contain a pol IIpromoter, as described herein. Examples of pol II promoters includeregulatable promoters and constitutive promoters. For example, thepromoter may be a CMV or RSV promoter. The expression cassette mayfurther contain a polyadenylation signal, such as a synthetic minimalpolyadenylation signal. The nucleic acid sequence may further contain amarker gene or stuffer sequences. The expression cassette may becontained in a viral vector. An appropriate viral vector for use in thepresent invention may be an adenoviral, lentiviral, adeno-associatedviral (AAV), poliovirus, herpes simplex virus (HSV) or murineMaloney-based viral vector. The gene of interest may be a geneassociated with a condition amenable to siRNA therapy. Examples of suchconditions include neurodegenerative diseases, such as atrinucleotide-repeat disease (e.g., polyglutamine repeat disease).Examples of these diseases include Huntington's disease or severalspinocerebellar ataxias. Alternatively, the gene of interest may encodea ligand for a chemokine involved in the migration of a cancer cell, ora chemokine receptor.

The present invention also provides an expression cassette containing anisolated nucleic acid sequence encoding a first segment, a secondsegment located immediately 3′ of the first segment, and a third segmentlocated immediately 3′ of the second segment, wherein the first andthird segments are each less than 30 base pairs in length and each morethan 10 base pairs in length, and wherein the sequence of the thirdsegment is the complement of the sequence of the first segment, andwherein the isolated nucleic acid sequence functions as a smallinterfering RNA molecule (siRNA) targeted against a gene of interest.The expression cassette may be contained in a vector, such as a viralvector.

The present invention provides a method of reducing the expression of agene product in a cell by contacting a cell with an expression cassettedescribed above. It also provides a method of treating a patient byadministering to the patient a composition of the expression cassettedescribed above.

The present invention further provides a method of reducing theexpression of a gene product in a cell by contacting a cell with anexpression cassette containing an isolated nucleic acid sequenceencoding a first segment, a second segment located immediately 3′ of thefirst segment, and a third segment located immediately 3′ of the secondsegment, wherein the first and third segments are each less than 30 basepairs in length and each more than 10 base pairs in length, and whereinthe sequence of the third segment is the complement of the sequence ofthe first segment, and wherein the isolated nucleic acid sequencefunctions as a small interfering RNA molecule (siRNA) targeted against agene of interest.

The present method also provides a method of treating a patient, byadministering to the patient a composition containing an expressioncassette, wherein the expression cassette contains an isolated nucleicacid sequence encoding a first segment, a second segment locatedimmediately 3′ of the first segment, and a third segment locatedimmediately 3′ of the second segment, wherein the first and thirdsegments are each less than 30 bases in length and each more than 10bases in length, and wherein the sequence of the third segment is thecomplement of the sequence of the first segment, and wherein theisolated nucleic acid sequence functions as a small interfering RNAmolecule (siRNA) targeted against a gene of interest.

I. Small Interfering RNA (siRNA)

A “small interfering RNA” or “short interfering RNA” or “siRNA” or“short hairpin RNA” or “shRNA” is a RNA duplex of nucleotides that istargeted to a nucleic acid sequence of interest, for example, ataxin-1or huntingtin (htt). As used herein, the term “siRNA” is a generic termthat encompasses the subset of shRNAs. A “RNA duplex” refers to thestructure formed by the complementary pairing between two regions of aRNA molecule. siRNA is “targeted” to a gene in that the nucleotidesequence of the duplex portion of the siRNA is complementary to anucleotide sequence of the targeted gene. In certain embodiments, thesiRNAs are targeted to the sequence encoding ataxin-1 or huntingtin. Insome embodiments, the length of the duplex of siRNAs is less than 30base pairs. In some embodiments, the duplex can be 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairsin length. In some embodiments, the length of the duplex is 19 to 25base pairs in length. In certain embodiment, the length of the duplex is19 or 21 base pairs in length. The RNA duplex portion of the siRNA canbe part of a hairpin structure. In addition to the duplex portion, thehairpin structure may contain a loop portion positioned between the twosequences that form the duplex. The loop can vary in length. In someembodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides inlength. In certain embodiments, the loop is 9 nucleotides in length. Thehairpin structure can also contain 3′ or 5′ overhang portions. In someembodiments, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5nucleotides in length.

The siRNA can be encoded by a nucleic acid sequence, and the nucleicacid sequence can also include a promoter. The nucleic acid sequence canalso include a polyadenylation signal. In some embodiments, thepolyadenylation signal is a synthetic minimal polyadenylation signal.

“Knock-down,” “knock-down technology” refers to a technique of genesilencing in which the expression of a target gene is reduced ascompared to the gene expression prior to the introduction of the siRNA,which can lead to the inhibition of production of the target geneproduct. The term “reduced” is used herein to indicate that the targetgene expression is lowered by 1-100%. In other words, the amount of RNAavailable for translation into a polypeptide or protein is minimized.For example, the amount of protein may be reduced by 10, 20, 30, 40, 50,60, 70, 80, 90, 95, or 99%. In some embodiments, the expression isreduced by about 90% (i.e., only about 10% of the amount of protein isobserved a cell as compared to a cell where siRNA molecules have notbeen administered). Knock-down of gene expression can be directed by theuse of dsRNAs or siRNAs.

“RNA interference (RNAi)” is the process of sequence-specific,post-transcriptional gene silencing initiated by siRNA. During RNAi,siRNA induces degradation of target mRNA with consequentsequence-specific inhibition of gene expression. RNAi involving the useof siRNA has been successfully applied to knockdown the expression ofspecific genes in plants, D. melanogaster, C. elegans, trypanosomes,planaria, hydra, and several vertebrate species including the mouse. Fora review of the mechanisms proposed to mediate RNAi, please refer toBass et al., 2001 Elbashir, 2001a, 2001b, 2001c; or Brantl, 2002.

According to a method of the present invention, the expression ofhuntingtin or atxain-1 can be modified via RNAi. For example, theaccumulation of huntingtin or atxain-1 can be suppressed in a cell. Theterm “suppressing” refers to the diminution, reduction or elimination inthe number or amount of transcripts present in a particular cell. Forexample, the accumulation of mRNA encoding huntingtin or atxain-1 can besuppressed in a cell by RNA interference (RNAi), e.g., the gene issilenced by sequence-specific double-stranded RNA (dsRNA), which is alsocalled short interfering RNA (siRNA). These siRNAs can be two separateRNA molecules that have hybridized together, or they may be a singlehairpin wherein two portions of a RNA molecule have hybridized togetherto form a duplex.

A mutant protein refers to the protein encoded by a gene having amutation, e.g., a missense or nonsense mutation in one or both allelesof huntingtin or atxain-1. A mutant huntingtin or atxain-1 may bedisease-causing, i.e., may lead to a disease associated with thepresence of huntingtin or atxain-1 in an animal having either one or twomutant allele(s).

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, “gene” refers to a nucleic acid fragment that expressesmRNA, functional RNA, or specific protein, including regulatorysequences. “Genes” also include nonexpressed DNA segments that, forexample, form recognition sequences for other proteins. “Genes” can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters. An“allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome.

The term “nucleic acid” refers to deoxyribonucleic acid (DNA) orribonucleic acid (RNA) and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base that is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. A “nucleic acid fragment” is a portion of a givennucleic acid molecule.

A “nucleotide sequence” is a polymer of DNA or RNA that can be single-or double-stranded, optionally containing synthetic, non-natural oraltered nucleotide bases capable of incorporation into DNA or RNApolymers.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidfragment,” “nucleic acid sequence or segment,” or “polynucleotide” areused interchangeably and may also be used interchangeably with gene,cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleicacid compositions. In the context of the present invention, an“isolated” or “purified” DNA molecule or RNA molecule is a DNA moleculeor RNA molecule that exists apart from its native environment and istherefore not a product of nature. An isolated DNA molecule or RNAmolecule may exist in a purified form or may exist in a non-nativeenvironment such as, for example, a transgenic host cell. For example,an “isolated” or “purified” nucleic acid molecule or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. In one embodiment, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Fragments andvariants of the disclosed nucleotide sequences are also encompassed bythe present invention. By “fragment” or “portion” is meant a full lengthor less than full length of the nucleotide sequence.

“Naturally occurring,” “native,” or “wild-type” is used to describe anobject that can be found in nature as distinct from being artificiallyproduced. For example, a protein or nucleotide sequence present in anorganism (including a virus), which can be isolated from a source innature and that has not been intentionally modified by a person in thelaboratory, is naturally occurring.

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of molecular biology techniques, as, for example, withpolymerase chain reaction PCR) and hybridization techniques. Variantnucleotide sequences also include synthetically derived nucleotidesequences, such as those generated, for example, by using site-directedmutagenesis, which encode the native protein, as well as those thatencode a polypeptide having amino acid substitutions. Generally,nucleotide sequence variants of the invention will have at least 40%,50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequenceidentity to the native (endogenous) nucleotide sequence.

The term “chimeric” refers to a gene or DNA that contains 1) DNAsequences, including regulatory and coding sequences that are not foundtogether in nature or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may include regulatory sequencesand coding sequences that are derived from different sources, or includeregulatory sequences and coding sequences derived from the same source,but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation. Transgenes include, for example, DNA that is eitherheterologous or homologous to the DNA of a particular cell to betransformed. Additionally, transgenes may include native genes insertedinto a non-native organism, or chimeric genes.

The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism.

A “foreign” gene refers to a gene not normally found in the hostorganism that has been introduced by gene transfer.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences. Because of the degeneracy ofthe genetic code, a large number of functionally identical nucleic acidsencode any given polypeptide. For instance, the codons CGT, CGC, CGA,CGG, AGA and AGG all encode the amino acid arginine. Thus, at everyposition where an arginine is specified by a codon, the codon can bealtered to any of the corresponding codons described without alteringthe encoded protein. Such nucleic acid variations are “silentvariations,” which are one species of “conservatively modifiedvariations.” Every nucleic acid sequence described herein that encodes apolypeptide also describes every possible silent variation, except whereotherwise noted. One of skill in the art will recognize that each codonin a nucleic acid (except ATG, which is ordinarily the only codon formethionine) can be modified to yield a functionally identical moleculeby standard techniques. Accordingly, each “silent variation” of anucleic acid that encodes a polypeptide is implicit in each describedsequence.

“Recombinant DNA molecule” is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook andRussell (2001).

The terms “heterologous gene”, “heterologous DNA sequence”, “exogenousDNA sequence”, “heterologous RNA sequence”, “exogenous RNA sequence” or“heterologous nucleic acid” each refer to a sequence that eitheroriginates from a source foreign to the particular host cell, or is fromthe same source but is modified from its original or native form. Thus,a heterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA or RNA sequence. Thus, theterms refer to a DNA or RNA segment that is foreign or heterologous tothe cell, or homologous to the cell but in a position within the hostcell nucleic acid in which the element is not ordinarily found.Exogenous DNA segments are expressed to yield exogenous polypeptides.

A “homologous” DNA or RNA sequence is a sequence that is naturallyassociated with a host cell into which it is introduced.

“Wild-type” refers to the normal gene or organism found in nature.

“Genome” refers to the complete genetic material of an organism.

A “vector” is defined to include, inter alia, any viral vector, as wellas any plasmid, cosmid, phage or binary vector in double or singlestranded linear or circular form that may or may not be selftransmissible or mobilizable, and that can transform prokaryotic oreukaryotic host either by integration into the cellular genome or existextrachromosomally (e.g., autonomous replicating plasmid with an originof replication).

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, which may include a promoter operably linkedto the nucleotide sequence of interest that may be operably linked totermination signals. The coding region usually codes for a functionalRNA of interest, for example an siRNA. The expression cassette includingthe nucleotide sequence of interest may be chimeric. The expressioncassette may also be one that is naturally occurring but has beenobtained in a recombinant form useful for heterologous expression. Theexpression of the nucleotide sequence in the expression cassette may beunder the control of a constitutive promoter or of an regulatablepromoter that initiates transcription only when the host cell is exposedto some particular stimulus. In the case of a multicellular organism,the promoter can also be specific to a particular tissue or organ orstage of development.

Such expression cassettes can include a transcriptional initiationregion linked to a nucleotide sequence of interest. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the gene of interest to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselectable marker genes.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence. It may constitute an “uninterrupted codingsequence”, i.e., lacking an intron, such as in a cDNA, or it may includeone or more introns bounded by appropriate splice junctions. An “intron”is a sequence of RNA that is contained in the primary transcript but isremoved through cleavage and re-ligation of the RNA within the cell tocreate the mature mRNA that can be translated into a protein.

The term “open reading frame” (ORF) refers to the sequence betweentranslation initiation and termination codons of a coding sequence. Theterms “initiation codon” and “termination codon” refer to a unit ofthree adjacent nucleotides (a ‘codon’) in a coding sequence thatspecifies initiation and chain termination, respectively, of proteinsynthesis (mRNA translation).

“Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA,siRNA, or other RNA that may not be translated but yet has an effect onat least one cellular process.

The term “RNA transcript” or “transcript” refers to the productresulting from RNA polymerase catalyzed transcription of a DNA sequence.When the RNA transcript is a perfect complementary copy of the DNAsequence, it is referred to as the primary transcript or it may be a RNAsequence derived from posttranscriptional processing of the primarytranscript and is referred to as the mature RNA. “Messenger RNA” (mRNA)refers to the RNA that is without introns and that can be translatedinto protein by the cell. “cDNA” refers to a single- or adouble-stranded DNA that is complementary to and derived from mRNA.

“Regulatory sequences” are nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences include enhancers, promoters, translationleader sequences, introns, and polyadenylation signal sequences. Theyinclude natural and synthetic sequences as well as sequences that may bea combination of synthetic and natural sequences. As is noted above, theterm “suitable regulatory sequences” is not limited to promoters.However, some suitable regulatory sequences useful in the presentinvention will include, but are not limited to constitutive promoters,tissue-specific promoters, development-specific promoters, regulatablepromoters and viral promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., 1995).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and may include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5) to itscoding sequence, which directs and/or controls the expression of thecoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. “Promoter” includes aminimal promoter that is a short DNA sequence comprised of a TATA-boxand other sequences that serve to specify the site of transcriptioninitiation, to which regulatory elements are added for control ofexpression. “Promoter” also refers to a nucleotide sequence thatincludes a minimal promoter plus regulatory elements that is capable ofcontrolling the expression of a coding sequence or functional RNA. Thistype of promoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. It is capable of operating in both orientations (normal orflipped), and is capable of functioning even when moved either upstreamor downstream from the promoter. Both enhancers and other upstreampromoter elements bind sequence-specific DNA-binding proteins thatmediate their effects. Promoters may be derived in their entirety from anative gene, or be composed of different elements derived from differentpromoters found in nature, or even be comprised of synthetic DNAsegments. A promoter may also contain DNA sequences that are involved inthe binding of protein factors that control the effectiveness oftranscription initiation in response to physiological or developmentalconditions. Examples of promoters that may be used in the presentinvention include the mouse U6 RNA promoters, synthetic human H1RNApromoters, SV40, CMV, RSV, RNA polymerase II and RNA polymerase IIIpromoters.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one of thesequences is affected by another. For example, a regulatory DNA sequenceis said to be “operably linked to” or “associated with” a DNA sequencethat codes for an RNA or a polypeptide if the two sequences are situatedsuch that the regulatory DNA sequence affects expression of the codingDNA sequence (i.e., that the coding sequence or functional RNA is underthe transcriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, heterologous gene or nucleic acid segment, or atransgene in cells. For example, in the case of siRNA constructs,expression may refer to the transcription of the siRNA only. Inaddition, expression refers to the transcription and stable accumulationof sense (mRNA) or functional RNA. Expression may also refer to theproduction of protein.

“Altered levels” refers to the level of expression in transgenic cellsor organisms that differs from that of normal or untransformed cells ororganisms.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples include the 3′non-regulatory regions of genes encoding nopaline synthase and the smallsubunit of ribulose bisphosphate carboxylase.

“Translation stop fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

The terms “cis-acting sequence” and “cis-acting element” refer to DNA orRNA sequences whose functions require them to be on the same molecule.An example of a cis-acting sequence on the replicon is the viralreplication origin.

The terms “trans-acting sequence” and “trans-acting element” refer toDNA or RNA sequences whose function does not require them to be on thesame molecule.

“Chromosomally-integrated” refers to the integration of a foreign geneor nucleic acid construct into the host DNA by covalent bonds. Wheregenes are not “chromosomally integrated” they may be “transientlyexpressed.” Transient expression of a gene refers to the expression of agene that is not integrated into the host chromosome but functionsindependently, either as part of an autonomously replicating plasmid orexpression cassette, for example, or as part of another biologicalsystem such as a virus.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when thecumulative alignment score falls off by the quantity X from its maximumachieved value, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide sequences wouldoccur by chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, thedefault parameters of the respective programs (e.g. BLASTN fornucleotide sequences) can be used. The BLASTN program (for nucleotidesequences) uses as defaults a wordlength (W) of 11, an expectation (E)of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands.Alignment may also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide matches and an identical percent sequenceidentity when compared to the corresponding alignment generated by thepreferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid sequences makes reference to a specified percentage ofnucleotides in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window, as measuredby sequence comparison algorithms or by visual inspection.

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%,or 99% sequence identity, compared to a reference sequence using one ofthe alignment programs described using standard parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl (1984); Tm 81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. Tm is reduced by about 1°C. for each 1% of mismatching; thus, Tm, hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theTm can be decreased 10° C. Generally, stringent conditions are selectedto be about 5° C. lower than the thermal melting point (Tm) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point(Tm); moderately stringent conditions can utilize a hybridization and/orwash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm);low stringency conditions can utilize a hybridization and/or wash at 11,12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm).Using the equation, hybridization and wash compositions, and desired T,those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T of lessthan 45° C. (aqueous solution) or 32° C. (formamide solution), it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993). Generally, highly stringent hybridization andwash conditions are selected to be about 5° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthand pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook and Russell 2001,for a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Forshort nucleic acid sequences (e.g., about 10 to 50 nucleotides),stringent conditions typically involve salt concentrations of less thanabout 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration(or other salts) at pH 7.0 to 8.3, and the temperature is typically atleast about 30° C. Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Very stringent conditions are selected to beequal to the Tm for a particular nucleic acid molecule.

Very stringent conditions are selected to be equal to the Tm for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. A “host cell” is a cell that has been transformed, or iscapable of transformation, by an exogenous nucleic acid molecule. Hostcells containing the transformed nucleic acid fragments are referred toas “transgenic” cells.

“Transformed,” “transduced,” “transgenic” and “recombinant” refer to ahost cell into which a heterologous nucleic acid molecule has beenintroduced. As used herein the term “transfection” refers to thedelivery of DNA into eukaryotic (e.g., mammalian) cells. The term“transformation” is used herein to refer to delivery of DNA intoprokaryotic (e.g., E. coli) cells. The term “transduction” is usedherein to refer to infecting cells with viral particles. The nucleicacid molecule can be stably integrated into the genome generally knownin the art. Known methods of PCR include, but are not limited to,methods using paired primers, nested primers, single specific primers,degenerate primers, gene-specific primers, vector-specific primers,partially mismatched primers, and the like. For example, “transformed,”“transformant,” and “transgenic” cells have been through thetransformation process and contain a foreign gene integrated into theirchromosome. The term “untransformed” refers to normal cells that havenot been through the transformation process.

“Genetically altered cells” denotes cells which have been modified bythe introduction of recombinant or heterologous nucleic acids (e.g., oneor more DNA constructs or their RNA counterparts) and further includesthe progeny of such cells which retain part or all of such geneticmodification.

As used herein, the term “derived” or “directed to” with respect to anucleotide molecule means that the molecule has complementary sequenceidentity to a particular molecule of interest.

“Gene silencing” refers to the suppression of gene expression, e.g.,transgene, heterologous gene and/or endogenous gene expression. Genesilencing may be mediated through processes that affect transcriptionand/or through processes that affect post-transcriptional mechanisms. Insome embodiments, gene silencing occurs when siRNA initiates thedegradation of the mRNA of a gene of interest in a sequence-specificmanner via RNA interference (for a review, see Brantl, 2002). In someembodiments, gene silencing may be allele-specific. “Allele-specific”gene silencing refers to the specific silencing of one allele of a gene.

“Knock-down,” “knock-down technology” refers to a technique of genesilencing in which the expression of a target gene is reduced ascompared to the gene expression prior to the introduction of the siRNA,which can lead to the inhibition of production of the target geneproduct. The term “reduced” is used herein to indicate that the targetgene expression is lowered by 1-100%. For example, the expression may bereduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 99%.Knock-down of gene expression can be directed by the use of dsRNAs orsiRNAs. For example, “RNA interference (RNAi),” which can involve theuse of siRNA, has been successfully applied to knockdown the expressionof specific genes in plants, D. melanogaster, C. elegans, trypanosomes,planaria, hydra, and several vertebrate species including the mouse. Fora review of the mechanisms proposed to mediate RNAi, please refer toBass et al., 2001, Elbashir et al., 2001 or Brantl 2002.

“RNA interference (RNAi)” is the process of sequence-specific,post-transcriptional gene silencing initiated by siRNA. RNAi is seen ina number of organisms such as Drosophila, nematodes, fungi and plants,and is believed to be involved in anti-viral defense, modulation oftransposon activity, and regulation of gene expression. During RNAi,siRNA induces degradation of target mRNA with consequentsequence-specific inhibition of gene expression.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides that is targeted to a gene interest. A “RNAduplex” refers to the structure formed by the complementary pairingbetween two regions of a RNA molecule. siRNA is “targeted” to a gene inthat the nucleotide sequence of the duplex portion of the siRNA iscomplementary to a nucleotide sequence of the targeted gene. In someembodiments, the length of the duplex of siRNAs is less than 30nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotidesin length. In some embodiments, the length of the duplex is 19-25nucleotides in length. The RNA duplex portion of the siRNA can be partof a hairpin structure. In addition to the duplex portion, the hairpinstructure may contain a loop portion positioned between the twosequences that form the duplex. The loop can vary in length. In someembodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides inlength. The hairpin structure can also contain 3′ or 5′ overhangportions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0,1, 2, 3, 4 or 5 nucleotides in length. Examples of sbRNA specific forhuntingin are encoded by the DNA sequences provided in FIG. 20. The“sense” and “antisense” sequences can be used with or without the loopregion indicated to form siRNA molecules. Other loop regions can besubstituted for the examples provided in this chart. As used herein, theterm siRNA is meant to be equivalent to other terms used to describenucleic acid molecules that are capable of mediating sequence specificRNAi, for example, double-stranded RNA (dsRNA), micro-RNA (miRNA), shorthairpin RNA (shRNA), short interfering oligonucleotide, shortinterfering nucleic acid, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetic silencing. For example, siRNAmolecules of the invention can be used to epigenetically silence genesat both the post-transcriptional level or the pre-transcriptional level.In a non-limiting example, epigenetic modulation of gene expression bysiRNA molecules of the invention can result from siRNA mediatedmodification of chromatin structure or methylation pattern to alter geneexpression (see, for example, Verdel et al., 2004, Science, 303,672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002,Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837;Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science,297, 2232-2237). In another non-limiting example, modulation of geneexpression by siRNA molecules of the invention can result from siRNAmediated cleavage of RNA (either coding or non-coding RNA) via RISC, oralternately, translational inhibition as is known in the art.

The siRNA can be encoded by a nucleic acid sequence, and the nucleicacid sequence can also include a promoter. The nucleic acid sequence canalso include a polyadenylation signal. In some embodiments, thepolyadenylation signal is a synthetic minimal polyadenylation signal.

“Treating” as used herein refers to ameliorating at least one symptomof, curing and/or preventing the development of a disease or acondition.

“Neurological disease” and “neurological disorder” refer to bothhereditary and sporadic conditions that are characterized by nervoussystem dysfunction, and which may be associated with atrophy of theaffected central or peripheral nervous system structures, or loss offunction without atrophy. A neurological disease or disorder thatresults in atrophy is commonly called a “neurodegenerative disease” or“neurodegenerative disorder.” Neurodegenerative diseases and disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, and repeat expansion neurodegenerativediseases, e.g., diseases associated with expansions of trinucleotiderepeats such as polyglutamine (polyQ) repeat diseases, e.g.,Huntington's disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,SCA6, SCA7, and SCA17), spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA). An example of a neurologicaldisorder that does not appear to result in atrophy is DYT1 dystonia.

The siRNAs of the present invention can be generated by any method knownto the art, for example, by in vitro transcription, recombinantly, or bysynthetic means. In one example, the siRNAs can be generated in vitro byusing a recombinant enzyme, such as T7 RNA polymerase, and DNAoligonucleotide templates.

II. Nucleic Acid Molecules of the Invention

The terms “isolated and/or purified” refer to in vitro isolation of anucleic acid, e.g., a DNA or RNA molecule from its natural cellularenvironment, and from association with other components of the cell,such as nucleic acid or polypeptide, so that it can be sequenced,replicated, and/or expressed. For example, “isolated nucleic acid” maybe a DNA molecule containing less than 31 sequential nucleotides that istranscribed into an siRNA. Such an isolated siRNA may, for example, forma hairpin structure with a duplex 21 base pairs in length that iscomplementary or hybridizes to a sequence in a gene of interest, andremains stably bound under stringent conditions (as defined by methodswell known in the art, e.g., in Sambrook and Russell, 2001). Thus, theRNA or DNA is “isolated” in that it is free from at least onecontaminating nucleic acid with which it is normally associated in thenatural source of the RNA or DNA and is preferably substantially free ofany other mammalian RNA or DNA. The phrase “free from at least onecontaminating source nucleic acid with which it is normally associated”includes the case where the nucleic acid is reintroduced into the sourceor natural cell but is in a different chromosomal location or isotherwise flanked by nucleic acid sequences not normally found in thesource cell, e.g., in a vector or plasmid.

In addition to a DNA sequence encoding a siRNA, the nucleic acidmolecules of the invention include double-stranded interfering RNAmolecules, which are also useful to inhibit expression of a target gene.

As used herein, the term “recombinant nucleic acid”, e.g., “recombinantDNA sequence or segment” refers to a nucleic acid, e.g., to DNA, thathas been derived or isolated from any appropriate cellular source, thatmay be subsequently chemically altered in vitro, so that its sequence isnot naturally occurring, or corresponds to naturally occurring sequencesthat are not positioned as they would be positioned in a genome whichhas not been transformed with exogenous DNA. An example of preselectedDNA “derived” from a source, would be a DNA sequence that is identifiedas a useful fragment within a given organism, and which is thenchemically synthesized in essentially pure form. An example of such DNA“isolated” from a source would be a useful DNA sequence that is excisedor removed from said source by chemical means, e.g., by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering.

Thus, recovery or isolation of a given fragment of DNA from arestriction digest can employ separation of the digest on polyacrylamideor agarose gel by electrophoresis, identification of the fragment ofinterest by comparison of its mobility versus that of marker DNAfragments of known molecular weight, removal of the gel sectioncontaining the desired fragment, and separation of the gel from DNA.Therefore, “recombinant DNA” includes completely synthetic DNAsequences, semi-synthetic DNA sequences, DNA sequences isolated frombiological sources, and DNA sequences derived from RNA, as well asmixtures thereof.

Nucleic acid molecules having base substitutions (i.e., variants) areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the nucleic acid molecule.

Oligonucleotide-mediated mutagenesis is a method for preparingsubstitution variants. This technique is known in the art as describedby Adelman et al. (1983). Briefly, nucleic acid encoding a siRNA can bealtered by hybridizing an oligonucleotide encoding the desired mutationto a DNA template, where the template is the single-stranded form of aplasmid or bacteriophage containing the unaltered or native genesequence. After hybridization, a DNA polymerase is used to synthesize anentire second complementary strand of the template that will thusincorporate the oligonucleotide primer, and will code for the selectedalteration in the nucleic acid encoding siRNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art.

The DNA template can be generated by those vectors that are eitherderived from bacteriophage M13 vectors (the commercially available M13mp 18 and M13 mp 19 vectors are suitable), or those vectors that containa single-stranded phage origin of replication. Thus, the DNA that is tobe mutated may be inserted into one of these vectors to generatesingle-stranded template. Production of the single-stranded template isdescribed in Chapter 3 of Sambrook and Russell, 2001. Alternatively,single-stranded DNA template may be generated by denaturingdouble-stranded plasmid (or other) DNA using standard techniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of the DNA, and the other strand (the original template) encodesthe native, unaltered sequence of the DNA. This heteroduplex molecule isthen transformed into a suitable host cell, usually a prokaryote such asE. coli JM101. After the cells are grown, they are plated onto agaroseplates and screened using the oligonucleotide primer radiolabeled with32-phosphate to identify the bacterial colonies that contain the mutatedDNA. The mutated region is then removed and placed in an appropriatevector, generally an expression vector of the type typically employedfor transformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutations(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthiodeoxyribocytosine called dCTP-(*S) (which can be obtained from theAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(*S) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101.

There are well-established criteria for designing siRNAs (see, e.g.,Elbashire et al., 2001a, 2001b, 2001c). Details can be found in thewebsites of several commercial vendors such as Ambion, Dharmacon andOligoengine. However, since the mechanism for siRNAs suppressing geneexpression is not entirely understood and siRNAs selected from differentregions of the same gene do not work as equally effective, very often anumber of siRNAs have to be generated at the same time in order tocompare their effectiveness.

III. Expression Cassettes of the Invention

To prepare expression cassettes, the recombinant DNA sequence or segmentmay be circular or linear, double-stranded or single-stranded.Generally, the DNA sequence or segment is in the form of chimeric DNA,such as plasmid DNA or a vector that can also contain coding regionsflanked by control sequences that promote the expression of therecombinant DNA present in the resultant transformed cell.

A “chimeric” vector or expression cassette, as used herein, means avector or cassette including nucleic acid sequences from at least twodifferent species, or has a nucleic acid sequence from the same speciesthat is linked or associated in a manner that does not occur in the“native” or wild type of the species.

Aside from recombinant DNA sequences that serve as transcription unitsfor an RNA transcript, or portions thereof, a portion of the recombinantDNA may be untranscribed, serving a regulatory or a structural function.For example, the recombinant DNA may have a promoter that is active inmammalian cells.

Other elements functional in the host cells, such as introns, enhancers,polyadenylation sequences and the like, may also be a part of therecombinant DNA. Such elements may or may not be necessary for thefunction of the DNA, but may provide improved expression of the DNA byaffecting transcription, stability of the siRNA, or the like. Suchelements may be included in the DNA as desired to obtain the optimalperformance of the siRNA in the cell.

Control sequences are DNA sequences necessary for the expression of anoperably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotic cells, for example,include a promoter, and optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Operably linked nucleic acids are nucleic acids placed in a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, operably linked DNA sequences are DNA sequencesthat are linked are contiguous. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

The recombinant DNA to be introduced into the cells may contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother embodiments, the selectable marker may be carried on a separatepiece of DNA and used in a co-transfection procedure. Both selectablemarkers and reporter genes may be flanked with appropriate regulatorysequences to enable expression in the host cells. Useful selectablemarkers are known in the art and include, for example,antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes that encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes a proteinwhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. For example, reporter genes include thechloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli andthe luciferase gene from firefly Photinus pyralis. Expression of thereporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells.

The general methods for constructing recombinant DNA that can transfecttarget cells are well known to those skilled in the art, and the samecompositions and methods of construction may be utilized to produce theDNA useful herein. For example, Sambrook and Russell, infra, providessuitable methods of construction.

The recombinant DNA can be readily introduced into the host cells, e.g.,mammalian, bacterial, yeast or insect cells by transfection with anexpression vector composed of DNA encoding the siRNA by any procedureuseful for the introduction into a particular cell, e.g., physical orbiological methods, to yield a cell having the recombinant DNA stablyintegrated into its genome or existing as a episomal element, so thatthe DNA molecules, or sequences of the present invention are expressedby the host cell. Preferably, the DNA is introduced into host cells viaa vector. The host cell is preferably of eukaryotic origin, e.g., plant,mammalian, insect, yeast or fungal sources, but host cells ofnon-eukaryotic origin may also be employed.

Physical methods to introduce a preselected DNA into a host cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, and the like. Biological methods tointroduce the DNA of interest into a host cell include the use of DNAand RNA viral vectors. For mammalian gene therapy, as described hereinbelow, it is desirable to use an efficient means of inserting a copygene into the host genome. Viral vectors, and especially retroviralvectors, have become the most widely used method for inserting genesinto mammalian, e.g., human cells. Other viral vectors can be derivedfrom poxviruses, herpes simplex virus I, adenoviruses andadeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

As discussed above, a “transfected”, “or “transduced” host cell or cellline is one in which the genome has been altered or augmented by thepresence of at least one heterologous or recombinant nucleic acidsequence. The host cells of the present invention are typically producedby transfection with a DNA sequence in a plasmid expression vector, aviral expression vector, or as an isolated linear DNA sequence. Thetransfected DNA can become a chromosomally integrated recombinant DNAsequence, which is composed of sequence encoding the siRNA.

To confirm the presence of the recombinant DNA sequence in the hostcell, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

To detect and quantitate RNA produced from introduced recombinant DNAsegments, RT-PCR may be employed. In this application of PCR, it isfirst necessary to reverse transcribe RNA into DNA, using enzymes suchas reverse transcriptase, and then through the use of conventional PCRtechniques amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique demonstrates the presence of an RNAspecies and gives information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and only demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect the recombinantDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the peptide products of theintroduced recombinant DNA sequences or evaluating the phenotypicchanges brought about by the expression of the introduced recombinantDNA segment in the host cell.

The instant invention provides a cell expression system for expressingexogenous nucleic acid material in a mammalian recipient. The expressionsystem, also referred to as a “genetically modified cell”, comprises acell and an expression vector for expressing the exogenous nucleic acidmaterial. The genetically modified cells are suitable for administrationto a mammalian recipient, where they replace the endogenous cells of therecipient. Thus, the preferred genetically modified cells arenon-immortalized and are non-tumorigenic.

According to one embodiment, the cells are transfected or otherwisegenetically modified ex vivo. The cells are isolated from a mammal(preferably a human), nucleic acid introduced (i.e., transduced ortransfected in vitro) with a vector for expressing a heterologous (e.g.,recombinant) gene encoding the therapeutic agent, and then administeredto a mammalian recipient for delivery of the therapeutic agent in situ.The mammalian recipient may be a human and the cells to be modified areautologous cells, i.e., the cells are isolated from the mammalianrecipient.

According to another embodiment, the cells are transfected or transducedor otherwise genetically modified in vivo. The cells from the mammalianrecipient are transduced or transfected in vivo with a vector containingexogenous nucleic acid material for expressing a heterologous (e.g.,recombinant) gene encoding a therapeutic agent and the therapeutic agentis delivered in situ.

As used herein, “exogenous nucleic acid material” refers to a nucleicacid or an oligonucleotide, either natural or synthetic, which is notnaturally found in the cells; or if it is naturally found in the cells,is modified from its original or native form. Thus, “exogenous nucleicacid material” includes, for example, a non-naturally occurring nucleicacid that can be transcribed into an anti-sense RNA, a siRNA, as well asa “heterologous gene” (i.e., a gene encoding a protein that is notexpressed or is expressed at biologically insignificant levels in anaturally-occurring cell of the same type). To illustrate, a syntheticor natural gene encoding human erythropoietin (EPO) would be considered“exogenous nucleic acid material” with respect to human peritonealmesothelial cells since the latter cells do not naturally express EPO.Still another example of “exogenous nucleic acid material” is theintroduction of only part of a gene to create a recombinant gene, suchas combining an regulatable promoter with an endogenous coding sequencevia homologous recombination.

IV. Methods for Introducing the Expression Cassettes of the Inventioninto Cells

The condition amenable to gene inhibition therapy may be a prophylacticprocess, i.e., a process for preventing disease or an undesired medicalcondition. Thus, the instant invention embraces a system for deliveringsiRNA that has a prophylactic function (i.e., a prophylactic agent) tothe mammalian recipient.

The inhibitory nucleic acid material (e.g., an expression cassetteencoding siRNA directed to a gene of interest) can be introduced intothe cell ex vivo or in vivo by genetic transfer methods, such astransfection or transduction, to provide a genetically modified cell.Various expression vectors (i.e., vehicles for facilitating delivery ofexogenous nucleic acid into a target cell) are known to one of ordinaryskill in the art.

As used herein, “transfection of cells” refers to the acquisition by acell of new nucleic acid material by incorporation of added DNA. Thus,transfection refers to the insertion of nucleic acid into a cell usingphysical or chemical methods. Several transfection techniques are knownto those of ordinary skill in the art including calcium phosphate DNAco-precipitation, DEAE-dextran, electroporation, cationicliposome-mediated transfection, tungsten particle-facilitatedmicroparticle bombardment, and strontium phosphate DNA co-precipitation.

In contrast, “transduction of cells” refers to the process oftransferring nucleic acid into a cell using a DNA or RNA virus. A RNAvirus (i.e., a retrovirus) for transferring a nucleic acid into a cellis referred to herein as a transducing chimeric retrovirus. Exogenousnucleic acid material contained within the retrovirus is incorporatedinto the genome of the transduced cell. A cell that has been transducedwith a chimeric DNA virus (e.g., an adenovirus carrying a cDNA encodinga therapeutic agent), will not have the exogenous nucleic acid materialincorporated into its genome but will be capable of expressing theexogenous nucleic acid material that is retained extrachromosomallywithin the cell.

The exogenous nucleic acid material can include the nucleic acidencoding the siRNA together with a promoter to control transcription.The promoter characteristically has a specific nucleotide sequencenecessary to initiate transcription. The exogenous nucleic acid materialmay further include additional sequences (i.e., enhancers) required toobtain the desired gene transcription activity. For the purpose of thisdiscussion an “enhancer” is simply any non-translated DNA sequence thatworks with the coding sequence (in cis) to change the basaltranscription level dictated by the promoter. The exogenous nucleic acidmaterial may be introduced into the cell genome immediately downstreamfrom the promoter so that the promoter and coding sequence areoperatively linked so as to permit transcription of the coding sequence.An expression vector can include an exogenous promoter element tocontrol transcription of the inserted exogenous gene. Such exogenouspromoters include both constitutive and regulatable promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a nucleic acid sequence under thecontrol of a constitutive promoter is expressed under all conditions ofcell growth. Constitutive promoters include the promoters for thefollowing genes which encode certain constitutive or “housekeeping”functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolatereductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK),pyruvate kinase, phosphoglycerol mutase, the beta-actin promoter, andother constitutive promoters known to those of skill in the art. Inaddition, many viral promoters function constitutively in eukaryoticcells. These include: the early and late promoters of SV40; the longterminal repeats (LTRs) of Moloney Leukemia Virus and otherretroviruses; and the thymidine kinase promoter of Herpes Simplex Virus,among many others.

Nucleic acid sequences that are under the control of regulatablepromoters are expressed only or to a greater or lesser degree in thepresence of an inducing or repressing agent, (e.g., transcription undercontrol of the metallothionein promoter is greatly increased in presenceof certain metal ions). Regulatable promoters include responsiveelements (REs) that stimulate transcription when their inducing factorsare bound. For example, there are REs for serum factors, steroidhormones, retinoic acid, cyclic AMP, and tetracycline and doxycycline.Promoters containing a particular RE can be chosen in order to obtain anregulatable response and in some cases, the RE itself may be attached toa different promoter, thereby conferring regulatability to the encodednucleic acid sequence. Thus, by selecting the appropriate promoter(constitutive versus regulatable; strong versus weak), it is possible tocontrol both the existence and level of expression of a nucleic acidsequence in the genetically modified cell. If the nucleic acid sequenceis under the control of an regulatable promoter, delivery of thetherapeutic agent in situ is triggered by exposing the geneticallymodified cell in situ to conditions for permitting transcription of thenucleic acid sequence, e.g., by intraperitoneal injection of specificinducers of the regulatable promoters which control transcription of theagent. For example, in situ expression of a nucleic acid sequence underthe control of the metallothionein promoter in genetically modifiedcells is enhanced by contacting the genetically modified cells with asolution containing the appropriate (i.e., inducing) metal ions in situ.

Accordingly, the amount of siRNA generated in, situ is regulated bycontrolling such factors as the nature of the promoter used to directtranscription of the nucleic acid sequence, (i.e., whether the promoteris constitutive or regulatable, strong or weak) and the number of copiesof the exogenous nucleic acid sequence encoding a siRNA sequence thatare in the cell.

In one embodiment of the present invention, an expression cassette maycontain a pol II promoter that is operably linked to a nucleic acidsequence encoding a siRNA. Thus, the pol II promoter, i.e., a RNApolymerase II dependent promoter, initiates the transcription of thesiRNA. In another embodiment, the pol II promoter is regulatable.

A pol II promoter may be used in its entirety, or a portion or fragmentof the promoter sequence may be used in which the portion maintains thepromoter activity. As discussed herein, pol II promoters are known to askilled person in the art and include the promoter of anyprotein-encoding gene, e.g., an endogenously regulated gene or aconstitutively expressed gene. For example, the promoters of genesregulated by cellular physiological events, e.g., heat shock, oxygenlevels and/or carbon monoxide levels, e.g., in hypoxia, may be used inthe expression cassettes of the invention. In addition, the promoter ofany gene regulated by the presence of a pharmacological agent, e.g.,tetracycline and derivatives thereof, as well as heavy metal ions andhormones may be employed in the expression cassettes of the invention.In an embodiment of the invention, the pol II promoter can be the CMVpromoter or the RSV promoter. In another embodiment, the pol II promoteris the CMV promoter.

As discussed above, a pol II promoter of the invention may be onenaturally associated with an endogenously regulated gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. The pol II promoter of theexpression cassette can be, for example, the same pol II promoterdriving expression of the targeted gene of interest. Alternatively, thenucleic acid sequence encoding the siRNA may be placed under the controlof a recombinant or heterologous pol II promoter, which refers to apromoter that is not normally associated with the targeted gene'snatural environment. Such promoters include promoters isolated from anyeukaryotic cell, and promoters not “naturally occurring,” i.e.,containing different elements of different transcriptional regulatoryregions, and/or mutations that alter expression. In addition toproducing nucleic acid sequences of promoters synthetically, sequencesmay be produced using recombinant cloning and/or nucleic acidamplification technology, including PCR™, in connection with thecompositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat.No. 5,928,906, each incorporated herein by reference).

In one embodiment, a pol II promoter that effectively directs theexpression of the siRNA in the cell type, organelle, and organism chosenfor expression will be employed. Those of ordinary skill in the art ofmolecular biology generally know the use of promoters for proteinexpression, for example, see Sambrook and Russell (2001), incorporatedherein by reference. The promoters employed may be constitutive,tissue-specific, inducible, and/or useful under the appropriateconditions to direct high level expression of the introduced DNAsegment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The identity of tissue-specificpromoters, as well as assays to characterize their activity, is wellknown to those of ordinary skill in the art.

In addition to at least one promoter and at least one heterologousnucleic acid sequence encoding the siRNA, the expression vector mayinclude a selection gene, for example, a neomycin resistance gene, forfacilitating selection of cells that have been transfected or transducedwith the expression vector.

Cells can also be transfected with two or more expression vectors, atleast one vector containing the nucleic acid sequence(s) encoding thesiRNA(s), the other vector containing a selection gene. The selection ofa suitable promoter, enhancer, selection gene and/or signal sequence isdeemed to be within the scope of one of ordinary skill in the artwithout undue experimentation.

The following discussion is directed to various utilities of the instantinvention. For example, the instant invention has utility as anexpression system suitable for silencing the expression of gene(s) ofinterest.

The instant invention also provides methods for genetically modifyingcells of a mammalian recipient in vivo. According to one embodiment, themethod comprises introducing an expression vector for expressing a siRNAsequence in cells of the mammalian recipient in situ by, for example,injecting the vector into the recipient.

V. Delivery Vehicles for the Expression Cassettes of the Invention

Delivery of compounds into tissues and across the blood-brain barriercan be limited by the size and biochemical properties of the compounds.Currently, efficient delivery of compounds into cells in vivo can beachieved only when the molecules are small (usually less than 600Daltons). Gene transfer for the correction of inborn errors ofmetabolism and neurodegenerative diseases of the central nervous system(CNS), and for the treatment of cancer has been accomplished withrecombinant adenoviral vectors.

The selection and optimization of a particular expression vector forexpressing a specific siRNA in a cell can be accomplished by obtainingthe nucleic acid sequence of the siRNA, possibly with one or moreappropriate control regions (e.g., promoter, insertion sequence);preparing a vector construct comprising the vector into which isinserted the nucleic acid sequence encoding the siRNA; transfecting ortransducing cultured cells in vitro with the vector construct; anddetermining whether the siRNA is present in the cultured cells.

Vectors for cell gene therapy include viruses, such asreplication-deficient viruses (described in detail below). Exemplaryviral vectors are derived from Harvey Sarcoma virus, ROUS Sarcoma virus,(MPSV), Moloney murine leukemia virus and DNA viruses (e.g.,adenovirus).

Replication-deficient retroviruses are capable of directing synthesis ofall virion proteins, but are incapable of making infectious particles.Accordingly, these genetically altered retroviral expression vectorshave general utility for high-efficiency transduction of nucleic acidsequences in cultured cells, and specific utility for use in the methodof the present invention. Such retroviruses further have utility for theefficient transduction of nucleic acid sequences into cells in vivo.Retroviruses have been used extensively for transferring nucleic acidmaterial into cells. Protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous nucleicacid material into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with the viral particles) are well knownin the art.

An advantage of using retroviruses for gene therapy is that the virusesinsert the nucleic acid sequence encoding the siRNA into the host cellgenome, thereby permitting the nucleic acid sequence encoding the siRNAto be passed on to the progeny of the cell when it divides. Promotersequences in the LTR region have can enhance expression of an insertedcoding sequence in a variety of cell types. Some disadvantages of usinga retrovirus expression vector are (1) insertional mutagenesis, i.e.,the insertion of the nucleic acid sequence encoding the siRNA into anundesirable position in the target cell genome which, for example, leadsto unregulated cell growth and (2) the need for target cellproliferation in order for the nucleic acid sequence encoding the siRNAcarried by the vector to be integrated into the target genome.

Another viral candidate useful as an expression vector fortransformation of cells is the adenovirus, a double-stranded DNA virus.The adenovirus is infective in a wide range of cell types, including,for example, muscle and endothelial cells. Adenoviruses (Ad) aredouble-stranded linear DNA viruses with a 36 kb genome. Several featuresof adenovirus have made them useful as transgene delivery vehicles fortherapeutic applications, such as facilitating in vivo gene delivery.Recombinant adenovirus vectors have been shown to be capable ofefficient in situ gene transfer to parenchymal cells of various organs,including the lung, brain, pancreas, gallbladder, and liver. This hasallowed the use of these vectors in methods for treating inheritedgenetic diseases, such as cystic fibrosis, where vectors may bedelivered to a target organ. In addition, the ability of the adenovirusvector to accomplish in situ tumor transduction has allowed thedevelopment of a variety of anticancer gene therapy methods fornon-disseminated disease. In these methods, vector containment favorstumor cell-specific transduction.

Like the retrovirus, the adenovirus genome is adaptable for use as anexpression vector for gene therapy, i.e., by removing the geneticinformation that controls production of the virus itself. Because theadenovirus functions in an extrachromosomal fashion, the recombinantadenovirus does not have the theoretical problem of insertionalmutagenesis.

Several approaches traditionally have been used to generate therecombinant adenoviruses. One approach involves direct ligation ofrestriction endonuclease fragments containing a nucleic acid sequence ofinterest to portions of the adenoviral genome. Alternatively, thenucleic acid sequence of interest may be inserted into a defectiveadenovirus by homologous recombination results. The desired recombinantsare identified by screening individual plaques generated in a lawn ofcomplementation cells.

Most adenovirus vectors are based on the adenovirus type 5 (Ad5)backbone in which an expression cassette containing the nucleic acidsequence of interest has been introduced in place of the early region 1(E1) or early region 3 (E3). Viruses in which E1 has been deleted aredefective for replication and are propagated in human complementationcells (e.g., 293 or 911 cells), which supply the missing gene E1 and pIXin trans.

In one embodiment of the present invention, one will desire to generatesiRNA in a brain cell or brain tissue. A suitable vector for thisapplication is an FIV vector (Brooks et al. (2002); Alisky et al.(2000a)) or an AAV vector. For example, one may use AAV5 (Davidson etal. (2000); Alisky et al. (2000a)). Also, one may apply poliovirus(Bledsoe et al. (2000)) or HSV vectors (Alisky et al. (2000b)).

Application of siRNA is generally accomplished by transfection ofsynthetic siRNAs, in vitro synthesized RNAs, or plasmids expressingshort hairpin RNAs (shRNAs). More recently, viruses have been employedfor in vitro studies and to generate transgenic mouse knock-downs oftargeted genes (Hannon 2002, Rubinson 2003, Kunath 2003). Recombinantadenovirus, adeno-associated virus (AAV) and feline immunodeficiencyvirus (FIV) can be used to deliver genes in vitro and in vivo (Alisky2000, Davidson 2000, Brooks 2000). Each has its own advantages anddisadvantages (Davidson 2003). Adenoviruses are double stranded DNAviruses with large genomes (36 kb) and have been engineered by mylaboratory and others to accommodate expression cassettes in distinctregions. We used recombinant adenoviruses expressing siRNAs todemonstrate successful viral-mediated gene suppression in brain (Xia2002).

Adeno-associated viruses have encapsidated genomes, similar to Ad, butare smaller in size and packaging capacity (˜30 nm vs. ˜100 nm;packaging limit of ˜4.5 kb). AAV contain single stranded DNA genomes ofthe + or the − strand. Eight serotypes of AAV (1-8) have been studiedextensively, three of which have been evaluated in the brain (Davidson2000, Passini 2003, Skorupa 1999, Frisella 2001, Xiao 1997, During1998). An important consideration for the present application is thatAAV5 transduces striatal and cortical neurons, and is not associatedwith any known pathologies.

Adeno associated virus (AAV) is a small nonpathogenic virus of theparvoviridae family (for review see Muzyczka, N. 1992. Curr TopMicrobiol Immunol 158: 97-129; see also U.S. Pat. No. 6,468,524). AAV isdistinct from the other members of this family by its dependence upon ahelper virus for replication. In the absence of a helper virus, AAV mayintegrate in a locus specific manner into the q arm of chromosome 19(Kotin et al., (1990) Proc. Natl. Acad. Sci. (USA) 87: 2211-2215). Theapproximately 5 kb genome of AAV consists of one segment of singlestranded DNA of either plus or minus polarity. The ends of the genomeare short inverted terminal repeats which can fold into hairpinstructures and serve as the origin of viral DNA replication. Physically,the parvovirus virion is non-enveloped and its icosohedral capsid isapproximately 20 nm in diameter.

To-date seven serologically distinct AAVs have been identified and fivehave been isolated from humans or primates and are referred to as AAVtypes 1-5 (Arella et al Handbook of Parvoviruses. Vol. 1. ed. P.Tijssen. Boca Raton, Fla., CRC Press, 1990). The most extensivelystudied of these isolates is AAV type 2 (AAV2). The genome of AAV2 is4680 nucleotides in length and contains two open reading frames (ORFs).The left ORF encodes the non-structural Rep proteins, Rep40, Rep 52,Rep68 and Rep 78, which are involved in regulation of replication andtranscription in addition to the production of single-stranded progenygenomes. Furthermore, two of the Rep proteins have been associated withthe possible integration of AAV genomes into a region of the q arm ofhuman chromosome 19. Rep68/78 have also been shown to possess NTPbinding activity as well as DNA and RNA helicase activities. The Repproteins possess a nuclear localization signal as well as severalpotential phosphorylation sites. Mutation of one of these kinase sitesresulted in a loss of replication activity.

The ends of the genome are short inverted terminal repeats which havethe potential to fold into T-shaped hairpin structures that serve as theorigin of viral DNA replication. Within the ITR region two elements havebeen described which are central to the function of the ITR, a GAGCrepeat motif and the terminal resolution site (trs). The repeat motifhas been shown to bind Rep when the ITR is in either a linear or hairpinconformation. This binding serves to position Rep68/78 for cleavage atthe trs which occurs in a site- and strand-specific manner. In additionto their role in replication, these two elements appear to be central toviral integration. Contained within the chromosome 19 integration locusis a Rep binding site with an adjacent trs. These elements have beenshown to be functional and necessary for locus specific integration.

The AAV2 virion is a non-enveloped, icosohedral particle approximately25 nm in diameter, consisting of three related proteins referred to asVPI,2 and 3. The right ORF encodes the capsid proteins, VP1, VP2, andVP3. These proteins are found in a ratio of 1:1:10 respectively and areall derived from the right-hand ORF. The capsid proteins differ fromeach other by the use of alternative splicing and an unusual startcodon. Deletion analysis has shown that removal or alteration of VP1which is translated from an alternatively spliced message results in areduced yield of infections particles. Mutations within the VP3 codingregion result in the failure to produce any single-stranded progeny DNAor infectious particles.

The following features of AAV have made it an attractive vector for genetransfer. AAV vectors have been shown in vitro to stably integrate intothe cellular genome; possess a broad host range; transduce both dividingand non dividing cells in vitro and in vivo and maintain high levels ofexpression of the transduced genes. Viral particles are heat stable,resistant to solvents, detergents, changes in pH, temperature, and canbe concentrated on CsCl gradients. Integration of AAV provirus is notassociated with any long term negative effects on cell growth ordifferentiation. The ITRs have been shown to be the only cis elementsrequired for replication, packaging and integration and may contain somepromoter activities.

Further provided by this invention are chimeric viruses where AAV can becombined with herpes virus, herpes virus amplicons, baculovirus or otherviruses to achieve a desired tropism associated with another virus. Forexample, the AAV4 ITRs could be inserted in the herpes virus and cellscould be infected. Post-infection, the ITRs of AAV4 could be acted on byAAV4 rep provided in the system or in a separate vehicle to rescue AAV4from the genome. Therefore, the cellular tropism of the herpes simplexvirus can be combined with AAV4 rep mediated targeted integration. Otherviruses that could be utilized to construct chimeric viruses includelentivirus, retrovirus, pseudotyped retroviral vectors, and adenoviralvectors.

Also provided by this invention are variant AAV vectors. For example,the sequence of a native AAV, such as AAV5, can be modified atindividual nucleotides. The present invention includes native and mutantAAV vectors. The present invention further includes all AAV serotypes.

FIV is an enveloped virus with a strong safety profile in humans;individuals bitten or scratched by FIV-infected cats do not seroconvertand have not been reported to show any signs of disease. Like AAV, FIVprovides lasting transgene expression in mouse and nonhuman primateneurons (Brooks 2002, Lotery 2002), and transduction can be directed todifferent cell types by pseudotyping, the process of exchanging theviruses native envelope for an envelope from another virus (Kang 2002,Stein 2001).

Thus, as will be apparent to one of ordinary skill in the art, a varietyof suitable viral expression vectors are available for transferringexogenous nucleic acid material into cells. The selection of anappropriate expression vector to express a therapeutic agent for aparticular condition amenable to gene silencing therapy and theoptimization of the conditions for insertion of the selected expressionvector into the cell, are within the scope of one of ordinary skill inthe art without the need for undue experimentation.

In another embodiment, the expression vector is in the form of aplasmid, which is transferred into the target cells by one of a varietyof methods: physical (e.g., microinjection, electroporation, scrapeloading, microparticle bombardment) or by cellular uptake as a chemicalcomplex (e.g., calcium or strontium co-precipitation, complexation withlipid, complexation with ligand). Several commercial products areavailable for cationic liposome complexation including Lipofectin™(Gibco-BRL, Gaithersburg, Md.) and Transfectam™ (ProMega, Madison,Wis.). However, the efficiency of transfection by these methods ishighly dependent on the nature of the target cell and accordingly, theconditions for optimal transfection of nucleic acids into cells usingthe above-mentioned procedures must be optimized. Such optimization iswithin the scope of one of ordinary skill in the art without the needfor undue experimentation.

VI. Diseases and Conditions Amendable to the Methods of the Invention

In the certain embodiments of the present invention, a mammalianrecipient to an expression cassette of the invention has a conditionthat is amenable to gene silencing therapy. As used herein, “genesilencing therapy” refers to administration to the recipient exogenousnucleic acid material encoding a therapeutic siRNA and subsequentexpression of the administered nucleic acid material in situ. Thus, thephrase “condition amenable to siRNA therapy” embraces conditions such asgenetic diseases (i.e., a disease condition that is attributable to oneor more gene defects), acquired pathologies (i.e., a pathologicalcondition that is not attributable to an inborn defect), cancers,neurodegenerative diseases, e.g., trinucleotide repeat disorders, andprophylactic processes (i.e., prevention of a disease or of an undesiredmedical condition). A gene “associated with a condition” is a gene thatis either the cause, or is part of the cause, of the condition to betreated. Examples of such genes include genes associated with aneurodegenerative disease (e.g., a trinucleotide-repeat disease such asa disease associated with polyglutamine repeats, Huntington's disease,and several spinocerebellar ataxias), and genes encoding ligands forchemokines involved in the migration of a cancer cells, or chemokinereceptor. Also siRNA expressed from viral vectors may be used for invivo antiviral therapy using the vector systems described.

Accordingly, as used herein, the term “therapeutic siRNA” refers to anysiRNA that has a beneficial effect on the recipient. Thus, “therapeuticsiRNA” embraces both therapeutic and prophylactic siRNA.

Differences between alleles that are amenable to targeting by siRNAinclude disease-causing mutations as well as polymorphisms that are notthemselves mutations, but may be linked to a mutation or associated witha predisposition to a disease state. An example of a targetablepolymorphism that is not itself a mutation is the polymorphism in exon58 associated with Huntington's disease.

Single nucleotide polymorphisms comprise most of the genetic diversitybetween humans. The major risk factor for developing Alzheimer's diseaseis the presence of a particular polymorphism in the apolipoprotein Egene.

Single nucleotide polymorphisms comprise most of the genetic diversitybetween humans, and that many disease genes, including the HD gene inHuntington's disease, contain numerous single nucleotide or multiplenucleotide polymorphisms that could be separately targeted in one allelevs. the other. The major risk factor for developing Alzheimer's diseaseis the presence of a particular polymorphism in the apolipoprotein Egene.

A. Gene Defects

A number of diseases caused by gene defects have been identified. Forexample, this strategy can be applied to a major class of disablingneurological disorders. For example this strategy can be applied to thepolyglutamine diseases, as is demonstrated by the reduction ofpolyglutamine aggregation in cells following application of thestrategy. The neurodegenerative disease may be a trinucleotide-repeatdisease, such as a disease associated with polyglutamine repeats,including Huntington's disease, and several spinocerebellar ataxias.Additionally, this strategy can be applied to a non-degenerativeneurological disorder, such as DYT1 dystonia.

B. Acquired Pathologies

As used herein, “acquired pathology” refers to a disease or syndromemanifested by an abnormal physiological, biochemical, cellular,structural, or molecular biological state. For example, the diseasecould be a viral disease, such as hepatitis or AIDS.

C. Cancers

The condition amenable to gene silencing therapy alternatively can be agenetic disorder or an acquired pathology that is manifested by abnormalcell proliferation, e.g., cancer. According to this embodiment, theinstant invention is useful for silencing a gene involved in neoplasticactivity. The present invention can also be used to inhibitoverexpression of one or several genes. The present invention can beused to treat neuroblastoma, medulloblastoma, or glioblastoma.

VII. Dosages, Formulations and Routes of Administration of the Agents ofthe Invention

The agents of the invention are preferably administered so as to resultin a reduction in at least one symptom associated with a disease. Theamount administered will vary depending on various factors including,but not limited to, the composition chosen, the particular disease, theweight, the physical condition, and the age of the mammal, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by the clinician employing animal models or other testsystems, which are well known to the art.

Administration of siRNA may be accomplished through the administrationof the nucleic acid molecule encoding the siRNA (see, for example,Felgner et al., U.S. Pat. No. 5,580,859, Pardoll et al. 1995; Stevensonet al. 1995; Molling 1997; Donnelly et al. 1995; Yang et al. II;Abdallah et al. 1995). Pharmaceutical formulations, dosages and routesof administration for nucleic acids are generally disclosed, forexample, in Felgner et al., supra.

The present invention envisions treating a disease, for example, aneurodegenerative disease, in a mammal by the administration of anagent, e.g., a nucleic acid composition, an expression vector, or aviral particle of the invention. Administration of the therapeuticagents in accordance with the present invention may be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of the agents of the invention may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses. Both local and systemic administration is contemplated.

One or more suitable unit dosage forms having the therapeutic agent(s)of the invention, which, as discussed below, may optionally beformulated for sustained release (for example using microencapsulation,see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of whichare incorporated by reference herein), can be administered by a varietyof routes including parenteral, including by intravenous andintramuscular routes, as well as by direct injection into the diseasedtissue. For example, the therapeutic agent may be directly injected intothe brain. Alternatively the therapeutic agent may be introducedintrathecally for brain and spinal cord conditions. In another example,the therapeutic agent may be introduced intramuscularly for viruses thattraffic back to affected neurons from muscle, such as AAV, lentivirusand adenovirus. The formulations may, where appropriate, be convenientlypresented in discrete unit dosage forms and may be prepared by any ofthe methods well known to pharmacy. Such methods may include the step ofbringing into association the therapeutic agent with liquid carriers,solid matrices, semi-solid carriers, finely divided solid carriers orcombinations thereof, and then, if necessary, introducing or shaping theproduct into the desired delivery system.

When the therapeutic agents of the invention are prepared foradministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations include from 0.1 to 99.9% by weight of the formulation. A“pharmaceutically acceptable” is a carrier, diluent, excipient, and/orsalt that is compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules, as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. The therapeutic agents of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual aerosol dose of each dosage formneed not in itself constitute an effective amount for treating theparticular indication or disease since the necessary effective amountcan be reached by administration of a plurality of dosage units.Moreover, the effective amount may be achieved using less than the dosein the dosage form, either individually, or in a series ofadministrations.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Specific non-limiting examples of the carriersand/or diluents that are useful in the pharmaceutical formulations ofthe present invention include water and physiologically acceptablebuffered saline solutions such as phosphate buffered saline solutions pH7.0-8.0. saline solutions and water.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLE 1 siRNA-Mediated Silencing of Genes Using Viral Vectors

In this Example, it is shown that genes can be silenced in anallele-specific manner. It is also demonstrated that viral-mediateddelivery of siRNA can specifically reduce expression of targeted genesin various cell types, both in vitro and in vivo. This strategy was thenapplied to reduce expression of a neurotoxic polyglutamine diseaseprotein. The ability of viral vectors to transduce cells efficiently invivo, coupled with the efficacy of virally expressed siRNA shown here,extends the application of siRNA to viral-based therapies and in vivotargeting experiments that aim to define the function of specific genes.

Experimental Protocols

Generation of the expression cassettes and viral vectors. The modifiedCMV (mCMV) promoter was made by PCR amplification of CMV by primers5′-AAGGTACCAGATCTTAGTTATTAATAGTAATCAATTACGG-3′ (SEQ ID NO:1) and5′-GAATCGATGCATGCCTCGAGACGGTTCACTAAACCAGCTCTGC-3′ (SEQ ID NO:2) withpeGFPN1 plasmid (purchased from Clontech, Inc) as template. The mCMVproduct was cloned into the KpnI and ClaI sites of the adenoviralshuttle vector pAd5KnpA, and was named pmCMVknpA. To construct theminimal polyA cassette, the oligonucleotides,5′-CTAGAACTAGTAATAAAGGATCCTTTATTTTCATTGGATCCGTGTGTTGGTTTTTTGTGTGCGGCCGCG-3′ (SEQ ID NO:3) and5′-TCGACGCGGCCGCACACAAAAAACCAACACACGGATCCAATGAAAATAAAGGATCCTTTATTACTAGTT-3′ (SEQ ID NO:4), were used. Theoligonucleotides contain SpeI and SalI sites at the 5′ and 3′ ends,respectively. The synthesized polyA cassette was ligated into SpeI, SalIdigested pmCMVKnpA. The resultant shuttle plasmid, pmCMVmpA was used forconstruction of head-to-head 21bp hairpins of eGFP (bp 418 to 438),human β-glucuronidase (bp 649 to 669), mouse β-glucuronidase (bp 646 to666) or E. coli β-galactosidase (bp 1152-1172). The eGFP hairpins werealso cloned into the Ad shuttle plasmid containing the commerciallyavailable CMV promoter and polyA cassette from SV40 large T antigen(pCMVsiGFPx). Shuttle plasmids were co-transfected into HEK293 cellsalong with the adenovirus backbones for generation of full-length Adgenomes. Viruses were harvested 6-10 days after transfection andamplified and purified as described (Anderson 2000).

Northern blotting. Total RNA was isolated from HEK293 cells transfectedby plasmids or infected by adenoviruses using TRIZOL® Reagent(Invitrogen™ Life Technologies, Carlsbad, Calif.) according to themanufacturer's instruction. RNAs (30 μg) were separated byelectrophoresis on 15% (wt/vol) polyacrylamide-urea gels to detecttranscripts, or on 1% agarose-formaldehyde gel for target mRNAsanalysis. RNAs were transferred by electroblotting onto hybond N+membrane (Amersham Pharmacia Biotech). Blots were probed with³²P-labeled sense (5′-CACAAGCTGGAGTACAACTAC-3′ (SEQ ID NO:5)) orantisense (5′-GTACTTGTACTCCAGCTTTGTG-3′ (SEQ ID NO:6)) oligonucleotidesat 37° C. for 3 h for evaluation of siRNA transcripts, or probed fortarget mRNAs at 42° C. overnight. Blots were washed using standardmethods and exposed to film overnight. In vitro studies were performedin triplicate with a minimum of two repeats.

In vivo studies and tissue analyses. Mice were injected into the tailvein (n=10 per group) or into the brain (n=6 per group) as describedpreviously (Stein 1999) with the virus doses indicated. Animals weresacrificed at the noted times and tissues harvested and sections ortissue lysates evaluated for β-glucuronidase expression, eGFPfluorescence, or β-galactosidase activity using established methods (Xia2001). Total RNA was harvested from transduced liver using the methodsdescribed above.

Cell Lines. PC12 tet off cell lines (Clontech Inc., Palo Alto, Calif.)were stably transfected with a tetracycline regulatable plasmid intowhich was cloned GFPQ19 or GFPQ80 (Chai 1999a). For GFP-Q80, clones wereselected and clone 29 chosen for regulatable properties and inclusionformation. For GFP-Q19 clone 15 was selected for uniformity of GFPexpression following gene expression induction. In all studies 1.5 μg/mldox was used to repress transcription. All experiments were done intriplicate and were repeated 4 times.

Results and Discussion

To accomplish intracellular expression of siRNA, a 21-bp hairpinrepresenting sequences directed against eGFP was constructed, and itsability to reduce target gene expression in mammalian cells using twodistinct constructs was tested. Initially, the siRNA hairpin targetedagainst eGFP was placed under the control of the CMV promoter andcontained a full-length SV-40 polyadenylation (polyA) cassette(pCMVsiGFPx). In the second construct, the hairpin was juxtaposed almostimmediate to the CMV transcription start site (within 6 bp) and wasfollowed by a synthetic, minimal polyA cassette (FIG. 1A, pmCMVsiGFPmpA)(Experimental Protocols), because we reasoned that functional siRNAwould require minimal to no overhangs (Caplan 2001; Nykänen 2001).Co-transfection of pmCMVsiGFPmpA with pEGFPN1 (Clontech Inc) into HEK293cells markedly reduced eGFP fluorescence (FIG. 1C). pmCMVsiGFPmpAtransfection led to the production of an approximately 63 bp RNAspecific for eGFP (FIG. 1D), consistent with the predicted size of thesiGFP hairpin-containing transcript. Reduction of target mRNA and eGFPprotein expression was noted in pmCMVsiGFPmpA-transfected cells only(FIG. 1E, F). In contrast, eGFP RNA, protein and fluorescence levelsremained unchanged in cells transfected with pEGFPN1 and pCMVsiGFPx(FIG. 1E, G), pEGFPN1 and pCMVsiβglucmpA (FIG. 1E, F, H), or pEGFPN1 andpCMVsiβgalmpA, the latter expressing siRNA against E. coliβ-galactosidase (FIG. 1E). These data demonstrate the specificity of theexpressed siRNAs.

Constructs identical to pmCMVsiGFPmpA except that a spacer of 9, 12 and21 nucleotides was present between the transcription start site and the21 bp hairpin were also tested. In each case, there was no silencing ofeGFP expression (data not shown). Together the results indicate that thespacing of the hairpin immediate to the promoter can be important forfunctional target reduction, a fact supported by recent studies in MCF-7cells (Brummelkamp 2002).

Recombinant adenoviruses were generated from the siGFP (pmCMVsiGFPmpA)and siβgluc (pmCMVsiβglucmpA) plasmids (Xia 2001; Anderson 2000) to testthe hypothesis that virally expressed siRNA allows for diminished geneexpression of endogenous targets in vitro and in vivo. HeLa cells are ofhuman origin and contain moderate levels of the soluble lysosomal enzymeβ-glucuronidase. Infection of HeLa cells with viruses expressing siβgluccaused a specific reduction in human β-glucuronidase mRNA (FIG. 1I)leading to a 60% decrease in β-glucuronidase activity relative to siGFPor control cells (FIG. 1J). Optimization of siRNA sequences usingmethods to refine target mRNA accessible sequences (Lee 2002) couldimprove further the diminution of β-glucuronidase transcript and proteinlevels.

The results in FIG. 1 are consistent with earlier work demonstrating theability of synthetic 21-bp double stranded RNAs to reduce expression oftarget genes in mammalian cells following transfection, with theimportant difference that in the present studies the siRNA wassynthesized intracellularly from readily available promoter constructs.The data support the utility of regulatable, tissue or cell-specificpromoters for expression of siRNA when suitably modified for closejuxtaposition of the hairpin to the transcriptional start site andinclusion of the minimal polyA sequence containing cassette (see,Methods above).

To evaluate the ability of virally expressed siRNA to diminishtarget-gene expression in adult mouse tissues in vivo, transgenic miceexpressing eGFP (Okabe 1997) were injected into the striatal region ofthe brain with 1×10⁷ infectious units of recombinant adenovirus vectorsexpressing siGFP or control siβgluc. Viruses also contained a dsRedexpression cassette in a distant region of the virus for unequivocallocalization of the injection site. Brain sections evaluated 5 daysafter injection by fluorescence (FIG. 2A) or western blot assay (FIG.2B) demonstrated reduced eGFP expression. Decreased eGFP expression wasconfined to the injected hemisphere (FIG. 2B). The in vivo reduction ispromising, particularly since transgenically expressed eGFP is a stableprotein, making complete reduction in this short time frame unlikely.Moreover, evaluation of eGFP levels was done 5 days after injection,when inflammatory changes induced by the adenovirus vector likelyenhance transgenic eGFP expression from the CMV enhancer (Ooboshi 1997).

It was next tested whether virus mediated siRNA could decreaseexpression from endogenous alleles in vivo. Its ability to decreaseβ-glucuronidase activity in the murine liver, where endogenous levels ofthis relatively stable protein are high, was evaluated. Mice wereinjected via the tail vein with a construct expressing murine-specificsiβgluc (AdsiMuβgluc), or the control viruses Adsiβgluc (specific forhuman α-glucuronidase) or Adsiβgal. Adenoviruses injected into the tailvein transduced hepatocytes as shown previously (Stein 1999). Livertissue harvested 3 days later showed specific reduction of targetβ-glucuronidase RNA in AdsiMuβgluc treated mice only (FIG. 2C).Fluorometric enzyme assay of liver lysates confirmed these results, witha 12% decrease in activity from liver harvested from AdsiMuβglucinjected mice relative to Adsiβgal and Adsiβgluc treated ones (p<0.01;n=10). Interestingly, sequence differences between the murine and humansiRNA constructs are limited, with 14 of 21 bp being identical. Theseresults confirm the specificity of virus mediated siRNA, and indicatethat allele-specific applications are possible. Together, the data arethe first to demonstrate the utility of siRNA to diminish target geneexpression in brain and liver tissue in vivo, and establish thatallele-specific silencing in vivo is possible with siRNA.

One powerful therapeutic application of siRNA is to reduce expression oftoxic gene products in dominantly inherited diseases such as thepolyglutamine (polyQ) neurodegenerative disorders (Margolis 2001). Themolecular basis of polyQ diseases is a novel toxic property conferredupon the mutant protein by polyQ expansion. This toxic property isassociated with disease protein aggregation. The ability of virallyexpressed siRNA to diminish expanded polyQ protein expression in neuralPC-12 clonal cell lines was evaluated. Lines were developed that expresstetracycline-repressible eGFP-polyglutamine fusion proteins with normalor expanded glutamine of 19 (eGFP-Q19) and 80 (eGFP-Q80) repeats,respectively. Differentiated, eGFP-Q19-expressing PC12 neural cellsinfected with recombinant adenovirus expressing siGFP demonstrated aspecific and dose-dependent decrease in eGFP-Q19 fluorescence (FIG. 3A,C) and protein levels (FIG. 3B). Application of Adsiβgluc as a controlhad no effect (FIG. 3A-C). Quantitative image analysis of eGFPfluorescence demonstrated that siGFP reduced GFPQ19 expression bygreater than 96% and 93% for 100 and 50 MOI respectively, relative tocontrol siRNA (FIG. 3C). The multiplicity of infection (MOI) of 100required to achieve maximal inhibition of eGFP-Q19 expression resultslargely from the inability of PC12 cells to be infected byadenovirus-based vectors. This barrier can be overcome using AAV- orlentivirus-based expression systems (Davidson 2000; Brooks 2002).

To test the impact of siRNA on the size and number of aggregates formedin eGFP-Q80 expressing cells, differentiated PC-12/eGFP-Q80 neural cellswere infected with AdsiGFP or Adsiβgluc 3 days after doxycycline removalto induce GFP-Q80 expression. Cells were evaluated 3 days later. Inmock-infected control cells (FIG. 4A), aggregates were very large 6 daysafter induction as reported by others (Chai 1999a; Moulder 1999). Largeaggregates were also seen in cells infected with Adsiβgluc (FIG. 4B),AdsiGFPx, (FIG. 4C, siRNA expressed from the normal CMV promoter andcontaining the SV40 large T antigen polyadenylation cassette), orAdsiβgal (FIG. 4D). In contrast, polyQ aggregate formation wassignificantly reduced in AdsiGFP infected cells (FIG. 4E), with fewerand smaller inclusions and more diffuse eGFP fluorescence.AdsiGFP-mediated reduction in aggregated and monomeric GFP-Q80 wasverified by Western blot analysis (FIG. 4F), and quantitation ofcellular fluorescence (FIG. 4G). AdsiGFP caused a dramatic and specific,dose-dependent reduction in eGFP-Q80 expression (FIG. 4F, G).

It was found that transcripts expressed from the modified CMV promoterand containing the minimal polyA cassette were capable of reducing geneexpression in both plasmid and viral vector systems (FIGS. 1-4). Theplacement of the hairpin immediate to the transcription start site anduse of the minimal polyadenylation cassette was of critical importance.In plants and Drosophila, RNA interference is initiated by theATP-dependent, processive cleavage of long dsRNA into 21-25 bpdouble-stranded siRNA, followed by incorporation of siRNA into aRNA-induced silencing complex that recognizes and cleaves the target(Nykanen 2001; Zamore 2000; Bernstein 2001; Hamilton 1999; Hammond2000). Viral vectors expressing siRNA are useful in determining ifsimilar mechanisms are involved in target RNA cleavage in mammaliancells in vivo.

In summary, these data demonstrate that siRNA expressed from viralvectors in vitro and in vivo specifically reduce expression of stablyexpressed plasmids in cells, and endogenous transgenic targets in mice.Importantly, the application of virally expressed siRNA to varioustarget alleles in different cells and tissues in vitro and in vivo wasdemonstrated. Finally, the results show that it is possible to reducepolyglutamine protein levels in neurons, which is the cause of at leastnine inherited neurodegenerative diseases, with a corresponding decreasein disease protein aggregation. The ability of viral vectors based onadeno-associated virus (Davidson 2000) and lentiviruses (Brooks 2002) toefficiently transduce cells in the CNS, coupled with the effectivenessof virally-expressed siRNA demonstrated here, extends the application ofsiRNA to viral-based therapies and to basic research, includinginhibiting novel ESTs to define gene function.

EXAMPLE 2 siRNA Specific for Huntingtin's Disease

The present inventors have developed huntingtin siRNA focused on twotargets. One is non-allele specific (siHDexon2), the other is targetedto the exon 58 codon deletion, the only known common intragenicpolymorphism in linkage dysequilibirum with the disease mutation(Ambrose et al, 1994). Specifically, 92% of wild type huntingtin alleleshave four GAGs in exon 58, while 38% of HD patients have 3 GAGs in exon58. To assess a siRNA targeted to the intragenic polymorphism, PC6-3cells were transfected with a full-length huntingtin containing the exon58 deletion. Specifically, PC6-3 rat pheochromocytoma cells wereco-transfected with CMV-human Htt (37Qs) and U6 siRNA hairpin plasmids.Cell extracts were harvested 24 hours later and western blots wereperformed using 15 μg total protein extract. Primary antibody was ananti-huntingtin monoclonal antibody (MAB2166, Chemicon) that reacts withhuman, monkey, rat and mouse Htt proteins.

As seen in FIG. 5, the siRNA lead to silencing of the disease allele. Asa positive control, a non-allele specific siRNA targeted to exon 2 ofthe huntingtin gene was used. siRNA directed against GFP was used as anegative control. It was noted that siEx58# 2 functional. The sequencefor siEX58#2 is the following: 5′-AAGAGGAGGAGGCCGACGCCC-3′ (SEQ IDNO:90). siEX58#1 was only minimally functional.

EXAMPLE 3 siRNA Specific for SCA1

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited,progressive neurodegenerative disease caused by an expandedpolyglutamine tract in ataxin-1. SCA1 is one of at least nineneurodegenerative diseases caused by polyglutamine expansion, whichincludes Huntington's disease (HD) and several other ataxias (Orr 1993,Zoghbi 1995). SCA1 is characterized by progressive ataxia, cerebellaratrophy, and loss of cerebellar Purkinje cells and brainstem neurons. Afeature common to all polyglutamine diseases, and many otherneurodegenerative diseases, is the formation of intracellular aggregatescontaining the disease protein, molecular chaperones, and components ofthe ubiquitin-proteasome pathway (Orr 1993, Zoghbi 1995). In SCA1, as inmany other polyQ diseases, the inclusions are intranuclear (Skinner1997).

Disease allele expansion ranges from 44 to 82 glutamines in SCA1, withrepeat length inversely correlated to age of disease onset (Zoghbi1995). Work in Drosophila models and transgenic mice demonstrate thatthe expansion confers a toxic gain of function on ataxin-1(Fernandez-Funez 2000, Burright 1995, Klement 1998). Recent work hasalso shown that phosphorylation of serine 776 of ataxin-1 by AKT, butnot nuclear aggregation, is required for SCA1 pathogenesis (Emamian2003, Chen 2003). Together, work in these model organisms has identifiedmanipulation of molecular chaperones, or inhibition of AKTphosphorylation of ataxin-1, as potential therapeutic routes(Fernandez-Funez 2000, Emamian 2003, Cummings 1998). As yet, however,there is no effective therapy for SCA1 or the other dominantneurodegenerative diseases caused by polyglutamine expansion.

Inhibition of mutant allele expression provides a direct approach toSCA1 therapy. In past years, antisense- or ribozyme-based techniquesheld promise in culture systems, but proved difficult to translate toanimal models. More recently, gene silencing through RNA interference(RNAi) has emerged as a powerful method to reduce target gene expressionin cell culture and, importantly, in brain (Caplen 2002, Miller 2003,Xia 2002, Davidson 2004). In the present experiments, the inventorstested whether the introduction of viral vectors expressing shorthairpin RNAs (shRNAs) directed against the transgenic human mutantataxin-1 gene would reduce pathology and ataxia in a mouse SCA1 model.

Vector construction and in vitro screening. Different target sites (F1to F11) were made based on the 2.4 kb human ataxin-1 ORF (gene accessionnumber: X79204). Sites were as follows: F1, bp 144-64; F2 bp 576-96; F3,bp 679-99; F4, 1334-54; F5, bp 490-510; F6, bp 2250-70; F7, bp 18-38;F8, bp 863-82; F9, bp 1876-96; F10, bp574-94; F11, bp 670-90. E. coliβ-galactosidase (bp 1152-1172) was used as control shRNA. Hairpins withloops 5′-ACTAGT-3′ (SEQ ID NO:104), or 5′-CTTCCTGTCA-3′ (SEQ ID NO:105)from mir23, were cloned into vectors containing the human U6 promoter,or the modified CMV promoter, by a two-step method as previouslydescribed (Xia 2002).

Flag-tagged ataxin-1 with normal (30Q) or expanded (82Q) polyglutamineregions were cloned into the AAV shuttle plasmid for testing hairpinsilencing. Plasmids expressing hairpins and plasmids expressing ataxin-1were co-transfected into HEK 293 cells or PC6-3 cells (4:1 ratio,hairpin to target), and cells lysed 48 to 72 h later. Western blots withanti-Flag were done to assess ataxin-1 levels. Actin was used a loadingcontrol.

Quantitative RT-PCR. HEK293 cells were transfected (Lipofectamine-2000,Invitrogen) with shLacZ, shScaI.F10 (571-592, Sca1-shSCA1.F10,5′-GGACACAAGGCTGAGCAGCAG-3′ (SEQ ID NO:102)), or shScaI.F11 (595-615,HSca1-shSCA1.F11, 5′-CAGCAGCACCTCAGCAGGGCTGCAGGATTAGTCAACCACCTCAGCAGGGCT-3′ (SEQ ID NO:103)) and a human ScaI expression plasmid in 2:1,4:1, or 8:1 molar ratios of shRNA:ScaI. RNA was harvested 24 hourspost-transfection using Trizol reagent (Invitrogen). Following DNasetreatment (DNA-free, Ambion), random-primed, first-strand cDNA wasgenerated from 1 mg total RNA (Taqman Reverse Transcription Reagents,Applied Biosystems) according to the manufacturer's protocol. cDNA wasdiluted four-fold and then used as template for real-time PCR. TaqmanAssays were performed on an ABI Prism 7000 Sequence Detection Systemusing Taqman 2× Universal PCR Master Mix (Applied Biosystems) andApplied Biosystems Assays-on-Demand Taqman primers/probe sets specificfor human ScaI and mammalian rRNA. Relative gene expression wasdetermined using the relative standard curve method (Applied BiosystemsUser Bulletin #2). Human ScaI expression levels were normalized to rRNAlevels and all samples were calibrated to the shLacZ 8:1 sample.

AAV vectors. pAAVshLacZ and pAAVshSCA1 contain human U6 driven hairpinsand CMV-hrGFP-SV40 polyA expression cassettes cloned between two AAV2ITR sequences. Flanking the AAV provirus are left and right armsequences from the Baculovirus Autographa californica, which are used togenerate recombinant Bacmid DNA through homologous recombination in E.coli. Recombinant Baculovirus were generated as described in theBac-to-Bac Baculovirus Expression System (InVitrogen), and AAV virus waspurified as described in Urabe et al (Urabe 2002). AAV titers weredetermined by DNA slot blot using an hrGFP-specific radiolabeled probe.

AAV injections. Injections into cerebella were as described by Alisky etal. (Alisky 2000), except that injections were administered 1 mm lateralto the midline, with a total of 3 μl injected into three separate sites.Transduction was targeted to midline lobules IV/V, with transductionspreading anterior-posterior to lobules III and VI, respectively. Virustiters were ˜1×10¹² vector genomes/ml as assessed by Q-PCR.

Northern Analysis. Total RNA was isolated using TRIZOL® Reagent(InVitrogen™ Life Technologies, Carlsbad, Calif.) according to themanufacturer's instructions. RNAs (30 mg) were separated byelectrophoresis on 15% (wt/vol) polyacrylamide-urea gels to detecttranscripts. RNAs were transferred by electroblotting onto Hybond N+membranes (Amersham Pharmacia Biotech). Blots were probed with32P-labeled sense oligonucleotides at 36° C. for 3 h for evaluation oftranscripts. Blots were washed in 2×SSC twice for 15 min at 36° C. andexposed to film overnight (Miyagishi 2002).

Immunohistochemistry and quantitation. Mice were perfused and fixedovernight with 4% paraformaldehyde in 0.2M phosphate buffer (pH 7.4).Tissues were cryoprotected by immersion in 25% sucrose and frozen inO.C.T. compound (Sakura Finetek U.S.A. Inc, Torrance, Calif.). Sagittalcryostat sections (10 um) were cut and mounted onto gelatin-coatedslides. For calbindin staining, no unmasking procedure was used.Ataxin-1 staining was done as described (Skinner 1997). Sections wereanalyzed using a Leica DM RBE and images acquired with a SPOT RT cameraand associated software (Diagnostics Instruments, Sterling Heights,Mich.). Measurement of molecular layer thickness and quantitation ofPurkinje cells were done using BioQuant system software (R & MBiometrics, Nashville, Tenn.) (Williams 1988).

Rotarod analysis. The Rotarod (Ugo Basile Biological Research Apparatus,model 7650) was used for these studies. Five-week-old mice werehabituated on the rotarod for 4 min, and then tested for 4 consecutivedays, 4 trials per day (˜30 minutes rest between trial). Mice wereretested two weeks after intracerebellar injection, and every two weeksuntil sacrifice at 16 wks. Additional groups of animals were tested outto 20 weeks. For each trial, the rod was accelerated from 4 to 40 rpmover 5 min, then maintained at 40 rpm until trial completion. Latency tofall (or if they hung on or rotated for two consecutive rotationswithout running) was recorded for each mouse. Any mouse remaining on theapparatus for 500 sec. was removed and scored as 500 sec.

Results

Optimization of ataxin-1-Targeting shRNAs

To accomplish RNAi for ataxin-1, the inventors developed short hairpins(shRNA) directed to the human 2.4 kb ataxin-1 cDNA for primary screeningin vitro. Short hairpin RNA (shRNA)-expressing plasmids wereco-transfected into HEK 293 cells with ataxin-1 (FLAG-tagged) expressionplasmids. Candidate hairpin sequences expressed from pol III (human U6;hU6) and pol II (modified CMV; mCMV) (Xia 2002) promoters were tested.The initial screen of hairpins directed against ataxin-1 sequencesdispersed along the ataxin-1 cDNA (FIG. 6A) was unsuccessful regardlessof promoter (0 of 4 tested). An expanded evaluation identified twoconstructs (shSCA1.F10 and shSCA1.F11; 2 of 7 tested) that reduced RNAlevels up to 80% and ataxin-1 protein levels by 50-60% (FIG. 6B, 6C).Q-PCR analysis showed that shSCA1.F10- and shSCA1.F11-mediated silencingof the ataxin-1 transcript was dose dependent (FIG. 6C). To determine ifshSCA1s were functional in neural cells the inventors used PC6-3 cells,a PC-12 cell derivative that displays more uniform neuronal phenotypes(Pittman 1993). PC6-3 cells were transfected with AAV shuttle vectorsexpressing shSCA1.F10, shSCA1.F11, or control shRNAs, and silencing ofataxin-1 expression was assessed by western blot. Interestingly,mCMV-expressed shSCA1.F11 appeared more efficient than the sameconstruct expressed from the hU6 promoter (FIG. 6D).

A recent study by Kawasaki and colleagues (Kawasaki 2003) suggested thatone caveat of Pol III-based promoters for expressing shRNAs isinefficient export of transcripts to the cytoplasm. Replacement of theloop structure of their shRNAs with those derived from endogenouslyexpressed miRNAs improved nuclear export and gene silencing (Kawasaki2003). To test if similar modifications improved Pol III-directedexpression of shRNAs for ataxin-1 silencing, the loops of hairpins fromshSCA1.F10 and shSCA1.F11, (originally 5′-ACTAGT-3′ (SEQ ID NO:104)),were replaced with the loop from miR23 (5′-CTTCCTGTCA-3′ (SEQ IDNO:105); designated F10 ml). While there was no effect of the miRNA loopon CMV-shRNA-based silencing (not shown), miR23 loops improved thesilencing activity of Pol III-expressed shSCA1.F10 and shSCA1.F11against normal human ataxin-1 (FIG. 6E) and importantly, human ataxin-1with an 82Q expansion (FIG. 6F).

Effects of shSCA1 on Motor Coordination in SCA1 Transgenic Mice

The inventors next generated recombinant adeno-associated virus serotype1 (AAV1) expressing shSCA1.F10 ml and shSCA1.F11mi to evaluate hairpinefficacy in the transgenic mouse model of SCA1 (denoted AAVshSCA1.F10mior AAVshSCA1.F11mi). The virus was also engineered to express the hrGFPreporter for detection of transduced cells (FIG. 7A). In SCA1 mice,transgenic human disease allele (ataxin-1-Q82) expression is confined tothe cerebellar Purkinje cells by PCP-2, a Purkinje cell-specificpromoter (Burright 1995, Clark 1997). Thus the inventors initiallytested AAV1's ability to transduce Purkinje cells, since itstransduction profile in cerebella was unknown. As shown in FIG. 7B,AAVshSCA1 readily transduces Purkinje cells. Northern blot of RNAharvested from cerebella 10 days after viral injection also showed thatshRNAs are expressed in vivo (FIG. 7C). The fast expression kineticsfrom AAV1 is similar to AAV serotype 5, which also shows tropism forPurkinje cells (Alisky 2000).

Heterozygous SCA1 transgenic mice display many of the characteristics ofhuman SCA1, including progressive ataxia, Purkinje cell degeneration,and thinning of cerebellar molecular layers. The rotarod test for motorperformance is a valid indicator of the progressive ataxia; proper footplacement in response to a changing environment (i.e., the rotating rod)challenges the cerebellum. To determine the effects of AAVshSCA1, orAAVs expressing control hairpins (AAVshLacZ), on the ataxic phenotype,mice were analyzed for baseline rotarod performance, followed byinjection at 7 weeks of age with shRNA-expressing viruses into midlinecerebellar lobules. Rotorod analyses were repeated every two weeks untilsacrifice. Mock-transduced animals (saline injection) were alsoassessed. The data in FIG. 7D demonstrate that transduction with virusesexpressing shSCA1.F10 ml, but not shLacZ, significantly improves SCA1mice motor performance. Also of note is the observation that expressionof shSCA1.F10 ml did not negatively affect the rotarod performance ofwildtype mice, indicating that intracellular expression of shRNAs is notovertly toxic to Purkinje cells.

Improved Neuropathology in shSCA1-Expressing Purkinje Cells

The inventors next tested if the improved rotarod performance wasattributable to improvements in neuropathology. The progressivepathological changes in SCA1 transgenic mice have been wellcharacterized, and include intranuclear inclusions of ataxin-1, Purkinjecell dendritic pruning, Purkinje cell loss and concomitant thinning ofthe cerebellar molecular layer (Burright 1995).

Cerebellar lobules from SCA1 and wildtype mice injected with AAVshLacZor AAVshSCA1 were evaluated for hrGFP expression and calbindin stainingto assess if shSCA1 reduced the progressive thinning of the molecularlayer in SCA1 transgenic mice. FIG. 8A shows representative sectionsfrom virus-injected mice cerebella. The juxtaposition of untransducedregions (hrGFP−) to transduced ones (hrGFP+) allowed for directcomparisons of the effects of shSCA1. Calbindin staining remained robustin hrGFP+ molecular layers from SCA1 transgenic mice treated withAAVshSCA1, but was notably diminished in untransduced areas. HrGFP+molecular layers from SCA1 transgenic mice injected with AAVshLacZshowed reduced calbindin staining, indistinguishable from untransducedlayers. In wildtype mice injected with AAVshSCA1 (FIG. 8A), AAVshLacZ orsaline (not shown), calbindin staining was uniform in all regionsexamined. The data show that shSCA1-mediated improvements are confinedto transduced neurons.

Molecular layer widths were quantified in wildtype mice and SCA1transgenic mice treated with AAV. FIG. 8B confirms the morphologicalobservation that expression of shRNAs did not affect the molecularlayers of wildtype mice. The data also show that molecular layer widthsin hrGFP+ regions from shSCA1-treated SCA1 mice (162 μm±16) areindistinguishable from wildtype controls (untransduced, 158 μm±20;AAVshSCA1 treated, 156 μm±20), in contrast to the markedly thinnedmolecular layer in SCA1 mice given AAVshLacZ (109 μm±12), or mockinjected (109 μm±11).

The inventors next determined the effects of AAVshSCA1 on human ataxin-1expression and the formation of ataxin-1 nuclear inclusions. Incerebella from SCA1 mice harvested 1 week after injection ofAAVshSCA1.F10 or AAVshSCA1.F11, ataxin-1 immuno-reactivity was markedlyreduced in transduced (hrGFP+) relative to non-transduced (GFP−) cells(FIG. 9). There was no effect of transduction on ataxin-1 levels in mockor AAVshLacZ treated SCA1 mice.

Prior work in the Orr and Zoghbi laboratories (Clark 1997) establishedthat mutant ataxin-1 forms single intranuclear inclusions in 50% ofPurkinje cells at 16 weeks of age. In tissues from SCA1 mice harvested 9weeks after injection of saline or AAVshLacZ, ataxin-1immunofluorescence (IF) was robust and present throughout Purkinje cellnuclei. The inventors found punctate intranuclear inclusions in 49% ofcells (FIG. 10A left panels; FIG. 10B top panel), independent of theirtransduction status. In contrast, transduced (hrGFP+) cells fromAAVshSCA1 treated mice displayed greatly diminished ataxin-1 nuclearstaining, with complete resolution of inclusions in transduced cells(FIGS. 10A, 10B and FIG. 11).

Discussion

The present results demonstrate in vivo efficacy of RNAi and support theutility of RNAi gene therapy for SCA1 and other polyglutamineneurodegenerative diseases. In the SCA1 mouse model, cerebellar deliveryof AAV1 vectors expressing ataxin-1-targeting shRNAs reduced ataxin-1expression in Purkinje cells, improved motor performance and normalizedthe cerebellar pathology in transduced regions. In these studies, theinventors directed delivery to midline cerebellar lobules because oftheir importance in axial and gait coordination in mammals. In tissuesharvested 9 weeks after injection, the inventors found near 100%transduction of targeted lobules, with a transduction efficiency of5-10% of all cerebellar Purkinje cells. This supports that directedcorrection could have a major impact on human disease characteristics.

SCA1 mice show progressive neurodegenerative disease similar to SCA1patients. In recent work using an inducible mouse model of SCA1,reversal of disease phenotypes was more difficult as the diseaseprogressed, suggesting that earlier treatments will be more beneficial(Zu 2004). In the inducible SCA1 model, inhibition of mutant ataxin-1expression at week 12 led to rotarod performance improvements.

The intranuclear, ataxin-1 inclusions are characteristic of SCA1 patientbrain tissue and SCA1 mice cerebellar Purkinje cells (Burright 1995).The inventors found complete resolution of inclusions in transducedcells, which correlated with improved neuropathology. In the inducibleSCA1 model, inclusions resolved several days after inhibition of mutantallele expression. AAV1 expressed shRNAs reduced mutant ataxin-1expression as early as one week after introduction of vector, indicatingthat shSCA1-mediated inhibition of ataxin-1 (Q82) expression couldimprove disease-associated neuropathological changes almost immediatelyafter gene transfer.

In the inventors' initial in vitro screen, it was difficult to identifyeffective shRNAs for ataxin-1 silencing. The two functional shRNAsdiscovered by the inventors flanked the CAG repeat region. Thegeneralizability of this finding was tested in studies targeting amutant huntingtin and found that the CAG-repeat expansion in huntingtindid not confer accessibility to RNAi. Interestingly, shRNAs shSca1.F10and shSCA.F11 adhere less well to the model criteria (Reynolds 2004)than those that did not reduce ataxin-1 expression. This suggests thepotential requirement for screening many hairpins (perhaps up to 20)prior to identifying one suitably potent for gene silencing.

Heterozygous SCA1 mice provide a tool for allele-specific silencing ofthe disease gene; SCA1 mice retain two wildtype ataxin-1 genes inaddition to the human disease transgene. In SCA1 patients, however,shSCA1 would target both the disease and the wildtype allele. For SCA1this may not be problematic because ataxin-1 knock out mice do notdisplay cerebellar or brainstem pathology and have only mild ataxiameasured by rotarod performance. Moreover, shRNAs probably do not reducemRNA and protein levels to zero. The significant but non-ablativereduction of ataxin-1 would enable cellular machinery to ‘catch up’ withexistent inclusions.

In summary, the inventors have shown that RNAi therapy can dramaticallyimprove cellular and behavioral characteristics in a mouse model of ahuman dominant neurodegenerative disease, SCA1. The present findingshave relevance to other polyglutamine-repeat disorders includingHuntington's disease, and neurodegenerative disorders such asAlzheimer's disease, where inhibiting expression of a disease-linkedprotein would directly protect, or even reverse, disease phenotypes.

EXAMPLE 4 Huntington's Disease (HD)

Huntington's disease (HD) is one of several dominant neurodegenerativediseases that result from a similar toxic gain of function mutation inthe disease protein: expansion of a polyglutamine (polyQ)-encodingtract. It is well established that for HD and other polyglutaminediseases, the length of the expansion correlates inversely with age ofdisease onset. Animal models for HD have provided important clues as tohow mutant huntingtin (htt) induces pathogenesis. Currently, noneuroprotective treatment exists for HD. RNA interference has emerged asa leading candidate approach to reduce expression of disease genes bytargeting the encoding mRNA for degradation.

As discussed in Example 3 above, short hairpin RNAs (shRNAs) weregenerated that significantly inhibited human htt expression in celllines. Importantly, the shRNAs were designed to target sequences presentin HD transgenic mouse models. The present studies test the efficacy ofthe shRNAs in HD mouse models by determining if inclusions and otherpathological and behavioral characteristics that are representative ofHD can be inhibited or reversed. In a transgenic model of inducible HD,pathology and behavior improved when mutant gene expression was turnedoff. These experiments show that RNAi can prevent or reverse disease.

Although the effect of partial reduction of wildtype htt in adultneurons is unknown, it is advantageous to target only mutant htt fordegradation, if possible. One polymorphism in linkage disequilibriumwith HD has been identified in the coding sequence for htt, and othersare currently being investigated. Disease allele-specific RNAi aredesigned using approaches that led to allele specific silencing forother neurogenetic disease models. This would allow directed silencingof the mutant, disease-causing expanded allele, leaving the normalallele intact.

Constitutive expression of shRNA can prevent the neuropathological andbehavioral phenotypes in a mouse model of Spinocerebellar Ataxia type I,a related polyQ disease. However, the constitutive expression of shRNAmay not be necessary, particularly for pathologies that take many yearsto develop but may be cleared in a few weeks or months. For this reason,and to reduce long-term effects that may arise if nonspecific silencingor activation of interferon responses is noted, controlled expressionmay be very important. In order to regulate RNAi for diseaseapplication, doxycycline-responsive vectors have been developed forcontrolled silencing in vitro.

HD researchers benefit from a wealth of animal models including sixtransgenic and four knock-in mouse models (Bates 2003). Expression isfrom the endogenous human promoter, and the CAG expansion in the R6lines ranges from 110 to approximately 150 CAGs. The R6/2 line is themost extensively studied line from this work. R6/2 mice show aggressivedegenerative disease, with age of symptom onset at 8-12 weeks, and deathoccurring at 10 to 13 weeks. Neuronal intranuclear inclusions, ahallmark of HD patient brain, appear in the striatum and cortex of theR6/2 mouse (Meade 2002).

Adding two additional exons to the transgene and restricting expressionvia the prion promoter led to an HD mouse model displaying important HDcharacteristics but with less aggressive disease progression (Shilling1999, Shilling 2001). The Borchelt model, N171-82Q, has greater thanwildtype levels of RNA, but reduced amounts of mutant protein relativeto endogenous htt. N171-82Q mice show normal development for the first1-2 months, followed by failure to gain weight, progressiveincoordination, hypokinesis and tremors. There are statisticallysignificant differences in the rotarod test, alterations in gait, andhindlimb clasping. Mice show neuritic pathology characteristic of humanHD. Unlike the Bates model, there is limited neuronal loss.

Detloff and colleagues created a mouse knock-in model with an extensionof the endogenous mouse CAG repeat to approximately 150 CAGs. Thismodel, the CHL2 line, shows more aggressive phenotypes than prior mouseknock-in models containing few repeats (Lin 2001). Measurableneurological deficits include clasping, gait abnormalities, nuclearinclusions and astrogliosis.

The present studies utilize the well-characterized Borchelt mouse model(N171-82Q, line 81), and the Detloff knock-in model, the CHL2 line. Theinitial targets for htt silencing were focused on sequences present inthe N171-82Q transgene (exons 1-3). The use of this model wasadvantageous in the preliminary shRNA development because the RNAisearch could focus on only the amino-terminal encoding sequences ratherthan the full length 14 kb mRNA. FIG. 12 depicts the one-step cloningapproach used to screen hairpins (Harper 2004). No effective shRNAs werefound in exon 1, but several designed against exon 2, denoted shHDEx2.1(5′-AAGAAAGAACTTTCAGCTACC-3′, SEQ ID NO:96), shHDEx2.2 19 nt(5′-AGAACTTTCAGCTACCAAG-3′ (SEQ ID NO:97)), or shHDEx2.2 21 nt5′-AAAGAACTTTCAGCTACCAAG-3′ (SEQ ID NO:98)) and exon 3 (shHDEx3.1 19 nt5′-TGCCTCAACAAAGTTATCA-3′ (SEQ ID NO:99) or shHDEx3.1 21 nt5′-AATGCCTCAACAAAGTTATCA-3′ (SEQ ID NO:100)) sequences were effective.In co-transfection experiments with shRNA expressing plasmids and theN171-82Q transcript target, shHDEx2.1 reduced N171-Q82 transcript levelsby 80%, and protein expression by 60%.

In transient transfection assays shHDex2.1 did not silence a constructspanning exons 1-3 of mouse htt containing a 79 CAG repeat expansion,the mouse equivalent of N171-82Q. Next shHDEx2 into NIH 3T3 cells weretransfected to confirm that endogenous mouse htt, which is expressed inNIH 3T3 cells, would not be reduced. Surprisingly, shHDEx2.1 andshHDEx3.1 silenced full-length mouse htt. In contrast, shHDEx2.2silenced only the human N171-82Q transgene.

Yamamoto and colleagues and others have demonstrated that preformedinclusions can resolve (Yamamoto 2000). To test if RNAi could alsoreduce preformed aggregates, the inventors used a neuronal cell line,which, upon induction of Q80-eGFP expression, showed robust inclusionformation (Xia 2002). Cells laden with aggregates were mock-transduced,or transduced with recombinant virus expressing control shRNA, or shRNAsdirected against GFP. The inventors found dramatic reduction inaggregates as assessed by fluorescence. Quantification showed dosedependent effects (FIG. 13) that were corroborated by western blot (Xia2002).

As indicated in Example 1 above, viral vectors expressing siRNAs canmediate gene silencing in the CNS (Xia 2002). Also, as indicated inExample 3 above, these studies were extended to the mouse model ofspinocerebellar ataxia type 1 (SCA1). The data are important as theydemonstrate that shRNA is efficacious in the CNS of a mouse model ofhuman neurodegenerative disease. The data also support that shRNAexpression in brain is not detrimental to neuronal survival.

shRNAs can target the Exon 58 polymorphism. As described in Example 2above, a polymorphism in htt exon 58 is in linkage disequilibrium withHD (Ambrose 1994). Thirty eight percent of the HD population possesses a3-GAG repeat in exon 58, in contrast to the 4-GAG repeat found in 92% ofnon-HD patients. The polymorphism likely has no affect on htt, but itprovides a target for directing gene silencing to the disease allele. Asindicated in Example 2 above, in experiments to test if allele-specificsilencing for HD was possible, plasmids were generated that expressedshRNAs that were specific for the exon 58 polymorphism. The exon 583-GAG-targeting shRNAs were functional.

Developing vectors for control of RNAi in vivo. As demonstrated above,shRNA expressed from viral vectors is effective at directing genesilencing in brain. Also, viral vectors expressing shSCA1 inhibitedneurodegeneration in the SCA1 mouse model. ShRNA expression wasconstitutive in both instances. However, constitutive expression may notbe necessary, and could exacerbate any noted nonspecific effects. Thepresent inventors have developed and tested severaldoxycycline-regulated constructs. The construct depicted in FIG. 14showed strong suppression of target gene (GFP) expression after additionof doxycycline and RNAi induction.

RNAi can Protect, and/or Reverse, the Neuropathology in Mouse Models ofHuman Huntington's Disease

Two distinct but complimentary mouse models are used, the N171-82Qtransgenic and CHL2 knock-in mice. The former express a truncatedNH2-terminal fragment of human htt comprising exons 1-3 with an82Q-repeat expansion. The knock-in expresses a mutant mouse allele witha repeat size of ˜150. Neither shows significant striatal or corticalcell loss. Both therefore are suitable models for the early stages ofHD. They also possess similarities in mid- and end-stageneuropathological phenotypes including inclusions, gliosis, and motorand behavioral deficits that will permit comparison and validation. Onthe other hand, the differences inherent in the two models provideunique opportunities for addressing distinct questions regarding RNAitherapy. For example, N171-82Q transgenic mice have relatively earlydisease onset. Thus efficacy can be assessed within a few months, incontrast to 9 months or more in the CHL2 line. Because the data showedthat shHDEx2.2 targets the human transgene and not mouse HD, evaluatedisease-allele specific silencing in N171-82Q mice is evaluated. Incontrast, the CHL2 knock-in is important for testing how reducingexpression of both the mutant and wildtype alleles impacts on the HDphenotype. Finally, both models should be investigated because anytherapy for HD should be validated in two relevant disease models.

siRNA Against Human htt Protects Against Inclusion Formation in N171-82QMice

The data show that it is possible to silence the human N171-82Qtransgene in vitro, and work in reporter mice and SCA1 mouse modelsdemonstrated efficacy of RNAi in vivo in brain. shHDEx2.2 constructs,expressed from two vector systems with well-established efficacyprofiles in CNS, are now tested for their capacity to reduce mutanttransgenic allele expression in vivo. Further, the impact of shHDEx2.2on inclusion formation is assessed. Inclusions may not be pathogenicthemselves, but they are an important hallmark of HD and their presenceand abundance correlates with severity of disease in many studies.

Recombinant feline immunodeficiency virus (FIV) and adeno-associatedvirus (AAV) expressing shHDs are injected into N171-82Q. The levels ofshHDs expressed from FIV and AAV are evaluated, as is the ability toreduce htt mRNA and protein levels in brain, and subsequently affectinclusion formation.

Mice. N171-82Q mice developed by Borchelt and colleagues are used forthese experiments (Shilling 1999, Shilling 2001). The colony was set upfrom breeders purchased from Jackson Laboratories (N171-82Q, line 81)and are maintained as described (Shilling 1999, Shilling 2001). F1 pupsare genotyped by PCR off tail DNA, obtained when tagging weaned litters.

IC2 and EM48 have been used previously to evaluate N171-82Q transgeneexpression levels in brain by immuno-histochemistry (IHC) and westernblot (Zhou 2003, Trottier 1995). EM48 is an antibody raised against aGST-NH2 terminal fragment of htt that detects both ubiquitinated andnon-ubiquitinated htt-aggregates (Li 2000), and the IC2 antibodyrecognizes long polyglutamine tracts (Trottier 1995). By 4 weeksN171-82Q mice show diffuse EM48-positive staining in striata,hippocampus, cerebellar granule cells, and cortical layers IV and V(Shilling 1999, Shilling 2001). The present experiments focus on thestriatum and cortex because they are the major sites of pathology inhuman HD. TUNEL positivity and GFAP immunoreactivity are alsosignificant in striatal sections harvested from 3 month old N171-82Qmice (Yu 2003). At 4 months, punctate nuclear and cytoplasmicimmunoreactivity is also seen (Yu 2003).

Viruses. It is difficult to directly compare the two viruses under studyat equivalent doses; FIV is enveloped and can be concentrated andpurified, at best, to titers of 5×10⁸ infectious units/ml (iu/ml). FIVpseudotyped with the vesicular stomatitus glycoprotein (VSVg) are usedbecause of its tropism for neurons in the striatum (Brooks 2002). Incontrast, AAV is encapsidated and can be concentrated and purified totiters ranging from 1×10⁹ to 1×10¹¹ iu/ml, with 1×10¹⁰ titers onaverage. AAV serotype 5 is used because it is tropic for neurons instriatum and cortex, our target brain regions. Also, it diffuses widelyfrom the injection site (Alisky 2000, Davidson 2000). Ten-fold dilutionsof FIV and AAV generally results in a greater than 10-fold drop intransduction efficiency, making comparisons at equal titers, and doseescalation studies, unreasonable. Thus, both viruses are tested at thehighest titers routinely available to get a fair assessment of theircapacities for efficacy in N171-82Q mice. All viruses express thehumanized Renilla reniformis green fluorescent protein (hrGFP) reportertransgene in addition to the shRNA sequence (FIG. 15). This provides theunique opportunity to look at individual, transduced cells, and tocompare pathological improvements in transduced vs. untransduced cells.

Injections. Mice are placed into a David Kopf frame for injections. Miceare injected into the striatum (5 microliters; 100 nl/min) and thecortex (3 microliters; 75 nl/min) using a Hamilton syringe andprogrammable Harvard pump. The somatosensory cortex is targeted from aburr hole at −1.5 mm from Bregma, and 1.5 mm lateral. Depth is 0.5 mm.The striatum is targeted through a separate burr hole at +1.1 mm fromBregma, 1.5 mm lateral and 2 mm deep. Only the right side of the brainis injected, allowing the left hemisphere to be used as a control fortransgene expression levels and presence or absence of inclusions.

Briefly, groups of 4 week-old mice heterozygous for the N171-82Qtransgene and their age-matched wildtype littermates are injected withFIV (FIV groups are VSVg.FIV.shHDEx2.2, VSVg.FIVshlacZ, VSVg.hrGFP,saline) or AAV (AAV groups are AAV5.shHDEx2.2, AAV5shlacZ, AAV5 hrGFP,saline) (n=18/group; staggered injections because of the size of theexperiment). [Names of shHDEx2.2 and shlacZ expressing viruses have beenshortened from shlacZ.hrGFP, for example, to make it easier to read—butall vectors express hrGFP as reporter.] Nine mice/group are sacrificedat 12 weeks of age to assess the extent of transduction (eGFPfluorescence; viral copy number/brain region), shRNA expression(northern for shRNAs, and inhibition of expression of the transgenicallele (QPCR and western blot). The remaining groups are sacrificed at 5months of age. This experimental set up is repeated (to n=6/group) toconfirm results and test inter-experiment variability.

All mice in all groups are weighed bi-weekly (every other week) afterinitial weekly measurements. N171-82Q mice show normal weight gain up toapproximately 6 weeks, after which there are significant differenceswith their wildtype littermates.

PCR Analyses. Brains are harvested from mice sacrificed at 12 weeks ofage, and grossly evaluated for GFP expression to confirm transduction.The cortex and striatum from each hemisphere is dissected separately,snap frozen in liquid N2, pulverized with a mortar and pestle, andresuspended in Trizol (Gibco BRL). Separate aliquots are used forQ-RTPCR for N171-82Q transgenes and DNA PCR for viral genomes. Acoefficient of correlation is determined for transgene silencingrelative to viral genomes for both vector systems, for the regionsanalyzed and compared to contralateral striata and mice injected withcontrol vectors or saline.

The RNA harvested is used to evaluate activation ofinterferon-responsive genes. Bridges et al (Bridges 2003) and Sledz andcolleagues (Sledz 2003) found activation of 2′5′ oligo(A) polymerase(OAS) in cell culture with siRNAs and shRNAs, the latter expressed fromlentivirus vectors. Gene expression changes are assessed using QPCR forOAS, Stat1, interferon-inducible transmembrane proteins 1 and 2 andprotein kinase R (PKR). PKR activation is an initial trigger of thesignaling cascade of the interferon response.

Protein analyses. A second set of 3 brains/group are harvested forprotein analysis. Regions of brains are micro dissected as describedabove, and after pulverization are resuspended in extraction buffer (50mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1mM BetaME, 1× complete protease inhibitor cocktail) for analysis bywestern blot. HrGFP expression are evaluated and correlated todiminished levels of soluble N171-82Q using anti-GFP and antibodies tothe NH2-terminal region of htt (EM48) or the polyglutamine tract (IC2).

Histology. Histology is done on the remaining animals. Mice are perfusedwith 2% paraformaldehyde in PBS, brains blocked to remove thecerebellum, post-fixed ON, and then cryoprotected in 30% sucrose. Fullcoronal sections (40 μm) of the entire cerebrum are obtained using aMicrotome (American Products Co #860 equipped with a Super Histo Freezefreezing stage). Briefly, every section is collected, and sections 1-6are placed into 6 successive wells of a 24-well plate. Every 400microns, two sections each of 10 microns are collected for Nissl and H&Estaining. The process is repeated.

EM-48 immuno-staining reveals diffuse nuclear accumulations in N171-82Qmice as early as 4 weeks of age. In 6 mo. old mice inclusions areextensive (Shilling 2001). The increase in cytoplasmic and nuclear EM48immuno-reactivity, and in EM48 immuno-reactive inclusions over timeallow quantitative comparisons between transduced and untransducedcells. Again, control values are obtained from mice injected withshlacZ-expressing vectors, saline injected mice, and wt mice. Thecontralateral region is used as another control, with care taken to keepin mind the possibility of retrograde and anterograde transport of virusfrom the injection site.

Quantitation of nuclear inclusions is done using BioQuant™ software inconjunction with a Leitz DM RBE upright microscope equipped with amotorized stage (Applied Scientific Instruments). Briefly, floatingsections are stained with anti-NeuN (AMCA secondary) and EM48 antibodies(rhodamine secondary) followed by mounting onto slides. The regions tobe analyzed are outlined, and threshold levels for EM48 immunoreactivityset using sections from control injected mice. A minimum of 50hrGFP-positive and hrGFP negative neurons cells are evaluated per slide(5 slides/mouse), and inclusion intensity measured (arbitrary units).This is done for both striata and cortices. To quantitate cytoplasmicinclusions, the striatum is outlined and total EM48 aggregate densitymeasured. Threshold values are again done using control hemispheres andcontrol injected mice.

Additional wells of sections are stained with anti-GFAP,anti-neurofilament, and the lectin GSA to assay for viral orviral+hairpin induced gliosis, neuritic changes, and microglialactivation, respectively. GFAP-stained brain sections from N171-82Q miceshow gliosis by 4 months (Yu 1998), although earlier time points havenot been reported.

Stereology. In a separate experiment on N171-82Q mice and wt mice,unbiased stereology using BioQuant™ software is done to assesstransduction efficiency. Stereology allows for an unbiased assessment ofefficiency of transduction (number of cells transduced/input). AAV5(AAV5 hrGFP, AAV5shHD.hrGFP) and FIV (VSVg.FIVhrGFP, VSVg.FIVshHD.hrGFP)transduction efficiency is compared in the striatum and somatosensorycortex in HD and wildtype mice, with n=5 each. Mice are harvested at 12and 20 weeks. The cerebrum is sectioned in its entirety and stored at−20° C. until analysis. Briefly, six weeks after gene transfer withVSVg.FIVhrGFP (n=3) or AAV5 hrGFP (n=3), every section of an HD mousecerebrum is mounted and an initial assessment of the required numbers ofsections and grid and dissector size done using the coefficient of error(as determined by Martheron's quadratic approximation formula) as aguide.

The 171-82Q HD mouse model has important neuropathological andbehavioral characteristics relevant to HD. Onset of disease occursearlier than HD knock-in or YAC transgenic models, allowing an initial,important assessment of the protective effects of RNAi on thedevelopment of neuropathology and dysfunctional behavior, withoutincurring extensive long term housing costs. Admittedly, disease onsetis slower and less aggressive than the R6/2 mice created by Bates andcolleagues (Mangiarini 1996), but the R6/2 line is difficult to maintainand disease is so severe that it may be less applicable and lesspredicative of efficacy in clinical trials.

N171-82Q mice (n=6/group) and age-matched littermates (n=6/group) are beweighed twice a month from 4 wks on, and baseline rotarod testsperformed at 5 and 7 weeks of age. Numbers of mice per group are asdescribed in Schilling et al (Shilling 1999) in which statisticallysignificant differences between N171-82Q and wildtype littermates weredescribed. At 7 weeks of age (after testing is complete), AAV(AAVshHDEx2.2, AAVshlacZ, AAVhrGFP, saline) or FIV (FIVshEx2.2,FIVshlacZ, FIVhrGFP, saline) is injected bilaterally into the striatumand cortex. Rotarod tests are repeated at 3-week intervals starting atage 9 weeks, until sacrifice at 6 months. The clasping behavior isassessed monthly starting at 3 months.

Behavioral testing. N171-82Q mice are given four behavioral tests, allof which are standard assays for progressive disease in HD mouse models.The tests allow comparisons of behavioral changes resulting from RNAi tothose incurred in HD mouse models given other experimental therapies.For example, HD mice given cystamine or creatine therapy showed delayedimpairments in rotarod performance, and in some cases delayed weightloss (Ferrante 2000, Dedeoglu 2002, Dedeogu 2003) In addition to therotarod, which is used to assay for motor performance and generalneurological dysfunction, the activity monitor allows assessment of thedocumented progressive hypoactivity in N171-82Q mice. The beam analysisis a second test of motor performance that has also been used in HD micemodels (Carter 1999). Clasping, a phenotype of generalized neurologicaldysfunction, is straightforward and takes little time. Claspingphenotypes were corrected in R. Hen's transgenic mice possessing aninducible mutant htt.

Accelerated rotarod. N171-82Q and age-matched littermates are habituatedto the rotarod at week 4, and 4 trials per day for 4 days done on week 5and 7, and every 3 weeks hence using previously described assays(Shilling 1999, Clark 1997) in use in the lab. Briefly, 10 min trialsare run on an Economex rotarod (Columbus Instruments) set to acceleratefrom 4 to 40 rpm over the course of the assay. Latency to fall isrecorded and averages/group determined and plotted. Based on prior work(Shilling 1999) 6 mice will give sufficient power to assesssignificance.

Clasping behavior. Normal mice splay their limbs when suspended, butmice with neurological deficits can exhibit the opposite, with fore andhind limbs crunched into the abdomen (clasping). All mice are suspendedand scored for clasping monthly. The clasp must be maintained for atleast 30 sec. to be scored positive.

Activity monitor. Most HD models demonstrate hypokinetic behavior,particularly later in the disease process. This can be measured inseveral ways. One of the simplest methods is to monitor home cageactivity with an infrared sensor (AB-system 4.0, Neurosci Co., LTD).Measurements are taken over 3 days with one day prior habituation to thetesting cage (standard 12-hour light/dark cycle). Activity monitoring isdone at 12, 17, and 20 and 23 weeks of age.

Beam walking. N171Q-82Q and age matched littermates are assayed formotor performance and coordination using a series of successively moredifficult beams en route to an enclosed safety platform. The assay is asdescribed by Carter et al (Carter 1999). Briefly, 1 meter-length beamsof 28, 17 or 11 mm diameter are placed 50 cm above the bench surface. Asupport stand and the enclosed goal box flank the ends. Mice are trainedon the 11 mm beam at 6 weeks of age over 4 days, with 3 trials per day.If mice can traverse the beam in <20 sec. trials are initiated. A trialis then run on each beam, largest to smallest, with a 60 sec cutoff/beamand one minute rest between beams. A second trial is run and the meanscores of the two trials evaluated.

RNAi cannot replace neurons; it only has the potential to protectnon-diseased neurons, or inhibit further progression of disease at apoint prior to cell death. N171-82Q mice do not show noticeable cellularloss, and is therefore an excellent model of early HD in humans. Thegeneral methodology is the similar to that described above, except thatthe viruses are injected at 4 months, when N171-82Q mice have measurablebehavioral dysfunction and inclusions. Animals are sacrificed at endstage disease or at 8 months, whichever comes first. Histology, RNA andprotein in harvested brains are analyzed as described above.

It is important to confirm the biological effects of virally expressedshHDs in a second mouse model, as it is with any therapy. The Detloffknock-in mouse (the CHL2 line, also notated as HdhCAGQ150) is used as asecond model of early HD disease phenotypes. These mice have a CAGexpansion of approximately 150 units, causing brain pathologies similarto HD including gliosis and neural inclusions in the cortex andstriatum. They also show progressive motor dysfunction and otherbehavioral manifestations including rotarod deficits, clasping, gaitabnormalities and hypoactivity.

Heterozygous CHL2 mice express the mutant and wildtype allele at roughlyequivalent levels, and shRNAs directed against mouse HD silence bothtranscripts. shmHDEx2.1 causes reductions in gene expression, but notcomplete silencing. Disease severity in mouse models is dependent onmutant hit levels and CAG repeat length.

The inventors created shmHDEx2 (shRNA for murine HD) directed against aregion in mouse exon 2 that reduces expression of the full-length mouseHdh transcript in vitro. Transduction of neurons withshmHDEx2-expressing viruses, and its impacts on neuropathologicalprogression, behavioral dysfunction and the appearance of EM48immuno-reactive inclusions in CHL2 mice is tested. shmHD- orshlacZ-expressing vectors in CHL2 and wildtype brain is tested. In thisexperiment, virus is injected into the striatum of wt or CHL2 mice(10/group) using the coordinates described above, at 3 months of age.Two months later mice are sacrificed and brains removed and processedfor RNA (n=5/group) and protein (n=5).

A second study tests the vectors in the Detloff model. Briefly, 15 miceper group are injected into the striatum and cortex at 3 months of agewith AAV (AAVshmHD, AAVshlacZ, AAVhrGFP, saline) or FIV (VSVg.FIV.shmHD,VSVg.FIVshlacZ, VSVg.FIhrGFP, saline) expressing the transgenesindicated. To assess the impact of RNAi, activity performed. The miceare sacrificed at 16-18 months of age and five brains/group areprocessed for histology and sections banked in 24-well tissue cultureplates. The remaining brains are processed for RNA (n=6) and proteinanalysis (n=5). Northern blots or western blots are required to analyzewildtype and mutant htt expression because the only distinguishingcharacteristic is size.

Development of Effective Allele-Specific siRNAs

Mutant htt leads to a toxic gain of function, and inhibiting expressionof the mutant allele has a profound impact on disease (Yamamoto 2000).Also, selectively targeting the disease allele would be desirable ifnon-disease allele silencing is deleterious. At the present time, thereis one documented disease linked polymorphism in exon 58 (Lin 2001).Most non-HD individuals have 4 GAGs in Hdh exon 58 while 38% of HDpatients have 3 GAGs. As described above, RNAi can be accomplishedagainst the 3-GAG repeat.

Prior work by the inventors showed the importance of using full-lengthtargets for testing putative shRNAs. In some cases, shRNAs would workagainst truncated, but not full-length targets, or vice-versa. Thus, itis imperative that testable, full-length constructs are made to confirmallele-specific silencing. The V5 and FLAG tags provide epitopes toevaluate silencing at the mRNA and protein levels. This is important asputative shRNAs may behave as miRNAs, leading to inhibition ofexpression but not message degradation.

Designing the siRNAs. Methods are known for designing siRNAs (Miller2003, Gonzalez-Alegre 2003, xia 2002, Kao 2003). Information is alsoknow about the importance of maintaining flexibility at the 5′ end ofthe antisense strand for loading of the appropriate antisense sequenceinto the RISC complex (Khvorova 2003 Schwarz 2003). DNA sequences aregenerated by PCR. This method allows the rapid generation of manycandidate shRNAs, and it is significantly cheaper than buying shRNAs.Also, the inserts can be cloned readily into our vector shuttle plasmidsfor generation of virus. The reverse primer is a long oligonucleotideencoding the antisense sequence, the loop, the sense sequence, and aportion of the human U6 promoter. The forward primer is specific to thetemplate in the PCR reaction. PCR products are cloned directly intopTOPO blunt from InVitrogen, plasmids transformed into DH5a, andbacteria plated onto Kanr plates (the PCR template is Ampr). Kanr clonesare picked and sequenced. Sequencing is done with an extended ‘hotstart’ to allow effective read-through of the hairpin. Correct clonesare transfected into cells along with plasmids expressing the target orcontrol sequence (HttEx58.GAG3V5 and HttEx58.GAG4FLAG, respectively) andsilencing evaluated by western blot. Reductions in target mRNA levelsare assayed by Q-RTPCR. The control for western loading is neomycinphosphotransferase or hrGFP, which are expressed in thetarget-containing plasmids and provide excellent internal controls fortransfection efficiency. The control for Q-RTPCR is HPRT.

Cell lines expressing targets with the identified polymorphism orcontrol wildtype sequences are created. Target gene expression are undercontrol of an inducible promoter. PC6-3, Tet repressor (TetR+) cells, aPC-12 derivative with a uniform neuronal phenotype (Xia 2002) are used.PC6-3 cells are transfected with plasmids expressing HDEx58GAG3V5(contains neo marker) and HDEx58GAG4FLG (contains puro marker), andG418+/puromycin+ positive clones selected and characterized fortranscript levels and htt-V5 or htt-Flag protein levels.

FIV vectors expressing the allele specific shRNAs are generated and usedto test silencing in the inducible cell lines. FIV vectors infect mostepithelial and neuronal cell lines with high efficiency and aretherefore useful for this purpose. They also efficiently infect PC6-3cells. AAV vectors are currently less effective in in vitro screeningbecause of poor transduction efficiency in many cultured cell lines.

Cells are transduced with 1 to 50 infectious units/cell in 24-welldishes, 3 days after induction of mutant gene expression. Cells areharvested 72 h after infection and the effects on HDEx58.GAG3V5 orHDEx58GAG4FLG expression monitored.

EXAMPLE 5 Micro RNAi-Therapy for Polyglutamine-Disease

Post-transcriptional gene silencing occurs when double stranded RNA(dsRNA) is introduced or naturally expressed in cells. RNA interference(RNAi) has been described in plants (quelling), nematodes, andDrosophila. This process serves at least two roles, one as an innatedefense mechanism, and another developmental (Waterhouse 2001 Fire 1999,Lau 2001, Lagos-Quintana 2001, Lee 2001). RNAi may regulatedevelopmental expression of genes via the processing of small,temporally expressed RNAs, also called microRNAs (Knight 2001, Grishok2001). Harnessing a cell's ability to respond specifically to smalldsRNAs for target mRNA degradation has been a major advance, allowingrapid evaluation of gene function (Gonczy 2000, Fire 1998, Kennerdell1998, Hannon 2002, Shi 2003, Sui 2002).

Most eukaryotes encode a substantial number of small noncoding RNAstermed micro RNAs (miRNAs) (Zeng 2003, Tijsterman 2004, Lee 2004, Pham2004). mir-30 is a 22-nucleotide human miRNA that can be naturallyprocessed from a longer transcript bearing the proposed miR-30 stem-loopprecursor. mir-30 can translationally inhibit an mRNA-bearing artificialtarget sites. The mir-30 precursor stem can be substituted with aheterologous stem, which can be processed to yield novel miRNAs and canblock the expression of endogenous mRNAs.

Huntington's disease (HD) and Spinocerebellar ataxia type I (SCA1) aretwo of a class of dominant, neurodegenerative diseases caused by apolyglutamine (polyQ) expansion. The mutation confers a toxic gain offunction to the protein, with polyQ length predictive of age of onsetand disease severity. There is no curative or preventative therapy forHD or SCA1, supporting the investigation of novel strategies. Asdescribed above, the inventors showed that gene silencing by RNAinterference (RNAi) can be achieved in vitro and in vivo by expressingshort hairpin RNAs (shRNAs) specific for mRNAs encoding ataxin-1 orhuntingtin. Currently, strong, constitutive po1III promoters (U6 and H1)are used to express shRNAs, which are subsequently processed intofunctional small interfering RNAs (siRNAs). However, strong,constitutive expression of shRNAs may be inappropriate for diseases thattake several decades to manifest. Moreover, high-level expression may beunnecessary for sustained benefit, and in some systems may induce anon-specific interferon response leading to global shut-down of geneexpression. The inventors therefore generated polIII-expressed microRNAs(miRNAs) as siRNA shuttles as an alternative strategy. Due to theirendogenous nature, miRNA backbones may prevent the induction of theinterferon response.

Using human mir-30 as a template, miRNA shuttles were designed that uponprocessing by dicer released siRNAs specific for ataxin-1. Briefly, theconstructs were made by cloning a promoter (such as an induciblepromoter) and an miRNA shuttle containing an embedded siRNA specific fora target sequence (such as ataxin-1) into a viral vector. By cloning theconstruct into a viral vector, the construct can be effectivelyintroduced in vivo using the methods described in the Examples above.Constructs containing po1II-expressed miRNA shuttles with embeddedataxin-1-specific siRNAs were co-transfected into cells with GFP-taggedataxin-1, and gene silencing was assessed by fluorescence microscopy andwestern analysis. Dramatic arid dose-dependent gene silencing relativeto non-specific miRNAs carrying control siRNAs was observed. Thispo1II-based expression system exploits the structure of known miRNAs andsupports tissue-specific as well as inducible siRNA expression, andthus, serves as a unique and powerful alternative to dominantneurodegenerative disease therapy by RNAi.

Briefly, the constructs were made by cloning a promoter (such as aninducible promoter) and an miRNA shuttle containing an embedded siRNAspecific for a target sequence (such as ataxin-1) into a viral vector.By cloning the construct into a viral vector, the construct can beeffectively introduced in vivo using the methods described in theExamples above.

EXAMPLE 6 siRNA Suppression of Genes Involved in MJD/SCA3 and FTDP-17

Modulation of gene expression by endogenous, noncoding RNAs isincreasingly appreciated to play a role in eukaryotic development,maintenance of chromatin structure and genomic integrity. Recently,techniques have been developed to trigger RNA interference (RNAi)against specific targets in mammalian cells by introducing exogenouslyproduced or intracellularly expressed siRNAs. These methods have provento be quick, inexpensive and effective for knockdown experiments invitro and in vivo. The ability to accomplish selective gene silencinghas led to the hypothesis that siRNAs might be employed to suppress geneexpression for therapeutic benefit.

Dominantly inherited diseases are ideal candidates for siRNA-basedtherapy. To explore the utility of siRNA in inherited human disorders,the inventors employed cellular models to test whether we could targetmutant alleles causing two classes of dominantly inherited, untreatableneurodegenerative diseases: polyglutamine (polyQ) neurodegeneration inMJD/SCA3 and frontotemporal dementia with parkinsonism linked tochromosome 17 (FTDP-17). The polyQ neurodegenerative disorders consistof at least nine diseases caused by CAG repeat expansions that encodepolyQ in the disease protein. PolyQ expansion confers a dominant toxicproperty on the mutant protein that is associated with aberrantaccumulation of the disease protein in neurons. In FTDP-17, Taumutations lead to the formation of neurofibrillary tangles accompaniedby neuronal dysfunction and degeneration. The precise mechanisms bywhich these mutant proteins cause neuronal injury are unknown, butconsiderable evidence suggests that the abnormal proteins themselvesinitiate the pathogenic process. Accordingly, eliminating expression ofthe mutant protein by siRNA or other means should, in principle, slow oreven prevent disease. However, because many dominant disease genes mayalso encode essential proteins, the inventors sought to developsiRNA-mediated approaches that selectively inactivate mutant alleleswhile allowing continued expression of the wild type protein.

Methods

siRNA Synthesis. In vitro siRNA synthesis was previously described(Donze 2000). Reactions were performed with desalted DNAoligonucleotides (IDT Coralville, Iowa) and the AmpliScribeT7 High YieldTranscription Kit (Epicentre Madison, Wis.). Yield was determined byabsorbance at 260 nm. Annealed siRNAs were assessed for double strandedcharacter by agarose gel (1% w/v) electrophoresis and ethidium bromidestaining. Note that for all siRNAs generated in this study the most 5′nucleotide in the targeted cDNA sequence is referred to as position 1and each subsequent nucleotide is numbered in ascending order from 5′ to3′.

Plasmid Construction. The human ataxin-3 cDNA was expanded to 166 CAG'sby PCR (Laccone 1999). PCR products were digested at BamHI and KpnIsites introduced during PCR and ligated into BglII and KpnI sites ofpEGFP-N1 (Clontech) resulting in full-length expanded ataxin-3 fused tothe N-terminus of EGFP. Untagged Ataxin-3-Q166 was constructed byligating a PpuMI-NotI ataxin-3 fragment (3′ of the CAG repeat) intoAtaxin-3-Q166-GFP cut with PpuMI and NotI to remove EGFP and replace thenormal ataxin-3 stop codon. Ataxin-3-Q28-GFP was generated as above frompcDNA3.1-ataxin-3-Q28. Constructs were sequence verified to ensure thatno PCR mutations were present. Expression was verified by Western blotwith anti-ataxin-3 (Paulson 1997) and GFP antibodies (MBL). Theconstruct encoding a flag tagged, 352 residue tau isoform was previouslydescribed (Leger 1994). The pEGFP-tau plasmid was constructed byligating the human tau cDNA into pEGFP-C2 (Clontech) and encodes tauwith EGFP fused to the amino terminus. The pEGFP-tauV337M plasmid wasderived using site-directed mutagenesis (QuikChange Kit, Stratagene) ofthe pEFGP-tau plasmid.

Cell Culture and Transfections. Culture of Cos-7 and HeLa cells has beendescribed (Chai 1999b). Transfections with plasmids and siRNA wereperformed using Lipofectamine Plus (LifeTechnologies) according to themanufacturer's instructions. For ataxin-3 expression 1.5 μg plasmid wastransfected with 5 μg in vitro synthesized siRNAs. For Tau experiments 1μg plasmid was transfected with 2.5 μg siRNA. For expression of hairpinsiRNA from the phU6 constructs, 1 μg ataxin-3 expression plasmid wastransfected with 4 μg phU6-siC10i or phU6-siG10i. Cos-7 cells infectedwith siRNA-expressing adenovirus were transfected with 0.5 μg of eachexpression plasmid.

Stably transfected, doxycycline-inducible cell lines were generated in asubclone of PC12 cells, PC6-3, because of its strong neuraldifferentiation properties (Pittman 19938). A PC6-3 clone stablyexpressing Tet repressor plasmid (provided by S. Strack, Univ. of Iowa),was transfected with pcDNA5/TO-ataxin-3(Q28) or pcDNA5/TO-ataxin-3(Q166)(Invitrogen). After selection in hygromycin, clones were characterizedby Western blot and immunofluorescence. Two clones,PC6-3-ataxin3(Q28)#33 and PC6-3-ataxin3(Q166)#41, were chosen because oftheir tightly inducible, robust expression of ataxin-3.

siRNA Plasmid and Viral Production. Plasmids expressing ataxin-3 shRNAswere generated by insertion of head-to-head 21 bp hairpins in phU6 thatcorresponded to siC10 and siG10 (Xia 2002).

Recombinant adenovirus expressing ataxin-3 specific shRNA were generatedfrom phU6-C10i (encoding C10 hairpin siRNA) and phU6si-G10i (encodingG10 hairpin siRNA) as previously described (Xia 2002, Anderson 2000).

Western Blotting and Immunofluorescence. Cos-7 cells expressing ataxin-3were harvested 24-48 hours after transfection (Chai 1999b). Stablytransfected, inducible cell lines were harvested 72 hours afterinfection with adenovirus. Lysates were assessed for ataxin-3 expressionby Western blot analysis as previously described (Chai 1999b), usingpolyclonal rabbit anti-ataxin-3 antisera at a 1:15,000 dilution or 1C2antibody specific for expanded polyQ tracts (Trottier 1995) at a 1:2,500dilution. Cells expressing Tau were harvested 24 hours aftertransfection. Protein was detected with an affinity purified polyclonalantibody to a human tau peptide (residues 12-24) at a 1:500 dilution.Anti-alpha-tubulin mouse monoclonal antibody (Sigma St. Louis, Mo.) wasused at a 1:10,000 dilution and GAPDH mouse monoclonal antibody (SigmaSt. Louis, Mo.) was used at a 1:1,000 dilution.

Immunofluorescence for ataxin-3 (Chai 1999b) was carried out using 1C2antibody (Chemicon International Temecula, Calif.) at 1:1,000 dilution48 hours after transfection. Flag-tagged, wild type tau was detectedusing mouse monoclonal antibody (Sigma St. Louis, Mo.) at 1:1,000dilution 24 hours after transfection. Both proteins were detected withrhodamine conjugated secondary antibody at a 1:1,000 dilution.

Fluorescent Imaging and Quantification. Fixed samples were observed witha Zeiss Axioplan fluorescence microscope. Digital images were collectedon separate red, green and blue fluorescence channels using a SPOTdigital camera. Images were assembled and overlaid using Adobe Photoshop6.0. Live cell images were collected with a Kodak MDS 290 digital cameramounted to an Olympus (Tokyo, Japan) CK40 inverted microscope.Fluorescence was quantitated by collecting 3 non-overlapping images perwell at low power (10×). Pixel count and intensity for each image wasdetermined using Bioquant Nova Prime software (BIOQUANT Image AnalysisCorporation). Background was subtracted by quantitation of images fromcells of equivalent density under identical fluorescent illumination.Mock transfected cells were used to assess background fluorescence forall experiments and were stained with appropriate primary and secondaryantibodies for simulated heterozygous experiments. Average fluorescenceis reported from 2 to 3 independent experiments. The mean of 2 to 3independent experiments for cells transfected with the indicatedexpression plasmid and siMiss was set at one. Errors bars depictvariation between experiments as standard error of the mean. Insimulated heterozygous experiments, a blinded observer scored cells witha positive fluorescence signal for expression of wild type, mutant orboth proteins in random fields at high power for two independentexperiments. More than 100 cells were scored in each experiment andreported as number of cells with co-expression divided by total numberof transfected cells.

Results

Direct Silencing of Expanded Alleles. The inventors first attemptedsuppression of mutant polyQ expression using siRNA complementary to theCAG repeat and immediately adjacent sequences to determine if theexpanded repeat differentially altered the susceptibility of the mutantallele to siRNA inhibition (FIG. 17). HeLa cells were transfected withvarious in vitro synthesized siRNAs (Danze 2002) and plasmids encodingnormal or expanded polyQ fused to red or green fluorescent protein,respectively (Q19-RFP and Q80-GFP) (FIG. 16A). In negative control cellstransfected with Q80-GFP, Q19-RFP and a mistargeted siRNA (siMiss),Q80-GFP formed aggregates (Onodera 1997) which recruited the normallydiffuse Q19-RFP (FIG. 16A). When the experiment was performed with siRNAtargeted to GFP as a positive control for allele specific silencing,Q80-GFP expression was nearly abolished while Q19-RFP continued to beexpressed as a diffusely distributed protein (FIG. 16A). When Q19-RFPand Q80-GFP were co-transfected with siRNA directly targeting the CAGrepeat (siCAG) (FIG. 16A) or an immediately adjacent 5′ region (data notshown), expression of both proteins was efficiently suppressed.

To test whether siRNA could selectively silence expression of afull-length polyQ disease protein, siRNAs were designed that target thetranscript encoding ataxin-3, the disease protein in Machado-JosephDisease, also known as Spinocerebellar Ataxia Type 3 (MJD/SCA3) (Zoghbi2000) (FIG. 16B). In transfected cells, siRNA directed against threeseparate regions—the CAG repeat, a distant 5′ site, or a site just 5′ tothe CAG repeat (siN′CAG)—resulted in efficient, but not allele-specific,suppression of ataxin-3 containing normal or expanded repeats (data notshown). Consistent with an earlier study using longer dsRNA (Caplen2002) the present results show that expanded CAG repeats and adjacentsequences, while accessible to RNAi, may not be preferential targets forsilencing.

Allele-specific Silencing of the Mutant PolyQ Gene in MJD/SCA3. Infurther efforts to selectively inactivate the mutant allele theinventors took advantage of a SNP in the MJD1 gene, a G to C transitionimmediately 3′ to the CAG repeat (G987C) (FIG. 16B). This SNP is inlinkage disequilibrium with the disease-causing expansion, in mostfamilies segregating perfectly with the disease allele. Worldwide, 70%of disease chromosomes carry the C variant (Gaspar 2001). The presentataxin-3 expression cassettes, which were generated from patients(Paulson 1997), contain the C variant in all expanded ataxin-3constructs and the G variant in all normal ataxin-3 constructs. To testwhether this G-C mismatch could be distinguished by siRNA, siRNAs weredesigned that included the last 2 CAG triplets of the repeat followed bythe C variant at position 7 (siC7) (FIG. 17 and FIG. 16B), resulting ina perfect match only for expanded alleles. Despite the presence of asingle mismatch to the wild type allele, siC7 strongly inhibitedexpression of both alleles (FIG. 16C,D). A second G-C mismatch was thenintroduced at position 8 such that the siRNA contained two mismatches ascompared to wild type and only one mismatch as compared to mutantalleles (siC7/8). The siC7/8 siRNA effectively suppressed mutantataxin-3 expression, reducing total fluorescence to an average 8.6% ofcontrol levels, with only modest effects on wild type ataxin-3 (average75.2% of control). siC7/8 also nearly eliminated the accumulation ofaggregated mutant ataxin-3, a pathological hallmark of disease (Chan2000) (FIG. 16D).

To optimize differential suppression, siRNAs were designed containing amore centrally placed mismatch. Because the center of the antisensestrand directs cleavage of target mRNA in the RNA Induced SilencingComplex (RISC) complex (Elbashir 2001c), it was reasoned that centralmismatches might more efficiently discriminate between wild type andmutant alleles. siRNAs were designed that place the C of the SNP atposition 10 (siC10), preceded by the final three triplets in the CAGrepeat (FIG. 17 and FIG. 16B). In transfected cells, siC10 causedallele-specific suppression of the mutant protein (FIG. 16C,D).Fluorescence from expanded Atx-3-Q166-GFP was dramatically reduced (7.4%of control levels), while fluorescence of Atx-3-Q28-GFP showed minimalchange (93.6% of control; FIG. 16C,D). Conversely, siRNA engineered tosuppress only the wild type allele (siG10) inhibited wild typeexpression with little effect on expression of the mutant allele (FIG.16C,D). Inclusion of three CAG repeats at the 5′ end of the siRNA didnot inhibit expression of Q19-GFP, Q80-GFP, or full-length ataxin-1-Q30proteins that are each encoded by CAG repeat containing transcripts(FIG. 18).

In the disease state, normal and mutant alleles are simultaneouslyexpressed. In plants and worms, activation of RNAi against onetranscript results in the spread of silencing signals to other targetsdue to RNA-dependent RNA polymerase (RDRP) activity primed by theintroduced RNA (Fire 1998, Tang 2003). Although spreading has not beendetected in mammalian cells and RDRP activity is not required foreffective siRNA inhibition (Chiu 2002, Schwarz 2002, Martinez 2002),most studies have used cell-free systems in which a mammalian RDRP couldhave been inactivated. If triggering the mammalian RNAi pathway againstone allele activates cellular mechanisms that also silence the otherallele, then siRNA applications might be limited to non-essential genes.To test this possibility, the heterozygous state was simulated byco-transfecting Atx-3-Q28-GFP and Atx-3-Q166 and analyzing suppressionby Western blot. As shown in FIG. 16E each siRNA retained thespecificity observed in separate transfections: siC7 inhibited bothalleles, siG10 inhibited only the wild type allele, and siC7/8 and siC10inhibited only mutant allele expression.

Effective siRNA therapy for late onset disease will likely requiresustained intracellular expression of the siRNA. Accordingly, thepresent experiments were extended to two intracellular methods of siRNAproduction and delivery: expression plasmids and recombinant virus(Brummelkamp 2002, Xia 2002). Plasmids were constructed expressing siG10or siC10 siRNA from the human U6 promoter as a hairpin transcript thatis processed intracellularly to produce siRNA (Brummelkamp 2002, Xia2002). When co-transfected with ataxin-3-GFP expression plasmids,phU6-G10i and phU6-C10i-siRNA plasmids specifically suppressed wild typeor mutant ataxin-3 expression, respectively (FIG. 16F).

This result encouraged the inventors to engineer recombinant adenoviralvectors expressing allele-specific siRNA (Xia 2002). Viral-mediatedsuppression was tested in Cos-7 cells transiently transfected with bothAtx-3-Q28-GFP and Atx-3-Q166 to simulate the heterozygous state. Cos-7cells infected with adenovirus encoding siG10, siC10 or negative controlsiRNA (Ad-G101, Ad-C10i, and Ad-LacZi respectively) exhibitedallele-specific silencing of wild type ataxin-3 expression with Ad-G10iand of mutant ataxin-3 with Ad-C100 (FIG. 19A,B,C). Quantitation offluorescence (FIG. 19B) showed that Ad-G100 reduced wild type ataxin-3to 5.4% of control levels while mutant ataxin-3 expression remainedunchanged. Conversely, Ad-C10i reduced mutant ataxin-3 fluorescencelevels to 8.8% of control and retained 97.4% of wild type signal. Theseresults were confirmed by Western blot where it was further observedthat Ad-G10i virus decreased endogenous (primate) ataxin-3 while Ad-C10idid not (FIG. 19C).

Viral mediated suppression was also assessed in differentiated PC 12neural cell lines that inducibly express normal (Q28) or expanded (Q166)mutant ataxin-3. Following infection with Ad-G10i, Ad-C10i, or Ad-LacZi,differentiated neural cells were placed in doxycycline for three days toinduce maximal expression of ataxin-3. Western blot analysis of celllysates confirmed that the Ad-G10i virus suppressed only wild typeataxin-3, Ad-C10i virus suppressed only mutant ataxin-3, and Ad-LacZihad no effect on either normal or mutant ataxin-3 expression (FIG. 19D).Thus, siRNA retains its efficacy and selectivity across different modesof production and delivery to achieve allele-specific silencing ofataxin-3.

Allele-Specific Silencing of a Missense Tau Mutation. The precedingresults indicate that, for DNA repeat mutations in which the repeatitself does not present an effective target, an associated SNP can beexploited to achieve allele-specific silencing. To test whether siRNAworks equally well to silence disease-causing mutations directly, theinventors targeted missense Tau mutations that cause FTDP-17 (Poorkaj1998, Hutton 1998). A series of 21-24 nt siRNAs were generated in vitroagainst four missense FTDP-17 mutations: G272V, P301L, V337M, and R406W(FIG. 17 and FIG. 20A). In each case the point mutation was placedcentrally, near the likely cleavage site in the RISC complex (position9, 10 or 11) (Laccone 1999). A fifth siRNA designed to target a 5′sequence in all Tau transcripts was also tested. To screen forsiRNA-mediated suppression, the inventors co-transfected GFP fusions ofmutant and wild type Tau isoforms together with siRNA into Cos-7 cells.Of the five targeted sites, the inventors obtained robust suppressionwith siRNA corresponding to V337M (FIG. 17 and FIG. 20A) (Poorkaj 1998,Hutton 1998), and thus focused further analysis on this mutation. TheV337M mutation is a G to A base change in the first position of thecodon (GTG to ATG), and the corresponding V337M siRNA contains the Amissense change at position 9 (siA9). This intended V337M-specific siRNApreferentially silenced the mutant allele but also caused significantsuppression of wild type Tau (FIG. 20B,C).

Based on the success of this approach with ataxin-3, the inventorsdesigned two additional siRNAs that contained the V337M (G to A)mutation at position 9 as well as a second introduced G-C mismatchimmediately 5′ to the mutation (siA9/C8) or three nucleotides 3′ to themutation (siA9/C12), such that the siRNA now contained two mismatches tothe wild type but only one to the mutant allele. This strategy resultedin further preferential inactivation of the mutant allele. One siRNA,siA9/C12, showed strong selectivity for the mutant tau allele, reducingfluorescence to 12.7% of control levels without detectable loss of wildtype Tau (FIG. 20B,C). Next, we simulated the heterozygous state byco-transfecting V337M-GFP and flag-tagged WT-Tau expression plasmids(FIG. 21). In co-transfected HeLa cells, siA9/C12 silenced the mutantallele (16.7% of control levels) with minimal alteration of wild typeexpression assessed by fluorescence (FIG. 21A) and Western blot (FIG.21B). In addition, siA9 and siA9/C8 displayed better allelediscrimination than we had observed in separate transfections, butcontinued to suppress both wild type and mutant tau expression (FIG.21A,B,C).

Discussion

Despite the rapidly growing siRNA literature, questions remainconcerning the design and application of siRNA both as a research tooland a therapeutic strategy. The present study, demonstratingallele-specific silencing of dominant disease genes, sheds light onimportant aspects of both applications.

Because many disease genes encode essential proteins, development ofstrategies to exclusively inactivate mutant alleles is important for thegeneral application of siRNA to dominant diseases. The present resultsfor two unrelated disease genes demonstrate that in mammalian cells itis possible to silence a single disease allele without activatingpathways analogous to those found in plants and worms that result in thespread of silencing signals (Fire 1998, Tang 2003).

In summary, siRNA can be engineered to silence expression of diseasealleles differing from wild type alleles by as little as a singlenucleotide. This approach can directly target missense mutations, as infrontotemporal dementia, or associated SNPs, as in MJD/SCA3. The presentstepwise strategy for optimizing allele-specific targeting extends theutility of siRNA to a wide range of dominant diseases in which thedisease gene normally plays an important or essential role. One suchexample is the polyglutamine disease, Huntington disease (HD), in whichnormal HD protein levels are developmentally essential (Nasir 1995). Theavailability of mouse models for many dominant disorders, includingMJD/SCA3 (Cemal 2002), HD (Lin 2001), and FTDP-17 (Tanemura 2002),allows for the in vivo testing of siRNA-based therapy for these andother human diseases.

EXAMPLE 7 Therapy for DYT1 Dystonia: Allele-Specific Silencing of MutantTorsinA

DYT1 dystonia is the most common cause of primary generalized dystonia.A dominantly inherited disorder, DYT1 usually presents in childhood asfocal dystonia that progresses to severe generalized disease. With onepossible exception, all cases of DYT1 result from a common GAG deletionin TOR1A, eliminating one of two adjacent glutamic acids near theC-terminus of the protein TorsinA (TA). Although the precise cellularfunction of TA is unknown, it seems clear that mutant TA (TAmut) actsthrough a dominant-negative or dominant-toxic mechanism. The dominantnature of the genetic defect in DYT1 dystonia suggests that efforts tosilence expression of TAmut should have potential therapeutic benefit.

Several characteristics of DYT1 make it an ideal disease in which toexplore siRNA-mediated gene silencing as potential therapy. Of greatestimportance, the dominant nature of the disease suggests that a reductionin mutant TA, whatever the precise pathogenic mechanism proves to be,will be helpful. Moreover, the existence of a single common mutationthat deletes a full three nucleotides suggests it may be feasible todesign siRNA that will specifically target the mutant allele and will beapplicable to all affected persons. Finally, there is no effectivetherapy for DYT1, a relentless and disabling disease. Thus, anytherapeutic approach with promise needs to be explored. Because TAwt maybe an essential protein, however, it is critically important thatefforts be made to silence only the mutant allele.

In the studies reported here, the inventors explored the utility ofsiRNA for DYT1. As outlined in the strategy in FIG. 22, the inventorssought to develop siRNA that would specifically eliminate production ofprotein from the mutant allele. By exploiting the three base pairdifference between wild type and mutant alleles, the inventorssuccessfully silenced expression of TAmut without interfering withexpression of the wild type protein (TAwt).

Methods

siRNA design and synthesis Small-interfering RNA duplexes weresynthesized in vitro according to a previously described protocol (Donze2002), using AmpliScribeT7 High Yield Transcription Kit (EpicentreTechnologies) and desalted DNA oligonucleotides (IDT). siRNAs weredesigned to target different regions of human TA transcript: 1) anupstream sequence common to both TAwt and TAmut (com-siRNA); 2) the areacorresponding to the mutation with either the wild type sequence(wt-siRNA) or the mutant sequence positioned at three different places(mutA-siRNA, mutB-siRNA, mutC-siRNA); and 3) a negative control siRNAcontaining an irrelevant sequence that does not target any region of TA(mis-siRNA). The design of the primers and targeted sequences are shownschematically in FIG. 23. After in vitro synthesis, the double strandedstructure of the resultant RNA was confirmed in 1.5% agarose gels andRNA concentration determined with a SmartSpect 3000 UV Spectrophotometer(BioRad).

Plasmids pcDNA3 containing TAwt or TAmut cDNA were kindly provided byXandra Breakefield (Mass General Hospital, Boston, Mass.). Thisconstruct was produced by cloning the entire coding sequences of humanTorsinA (1-332), both wild-type and mutant (GAG deleted), into themammalian expression vector, pcDNA3 (Clontech, Palo Alto, Calif.). UsingPCR based strategies, an N-terminal hemagglutinin (HA) epitope tag wasinserted into both constructs. pEGFP-C3-TAwt was kindly provided byPullanipally Shashidharan (Mt Sinai Medical School, NY). This constructwas made by inserting the full-length coding sequence of wild-typeTorsinA into the EcoRI and BamHI restriction sites of the vectorpEGFP-C3 (Clontech). This resulted in a fusion protein including eGFP,three “staffer” amino acids and the 331 amino acids of TorsinA.HA-tagged TAmut was inserted into the ApaI and SalI restriction sites ofpEGFP-C1 vector (Clontech), resulting in a GFP-HA-TAmut construct.

Cell culture and transfections Methods for cell culture of Cos-7 havebeen described previously (Chai 1999b). Transfections with DNA plasmidsand siRNA were performed using Lipofectamine Plus (LifeTechnologies)according to the manufacturer's instructions in six or 12 well plateswith cells at 70-90% confluence. For single plasmid transfection, 1 μgof plasmid was transfected with 5 μg of siRNA. For double plasmidtransfection, 0.75 μg of each plasmid was transfected with 3.75 μg ofsiRNA.

Western Blotting and Fluorescence Microscopy. Cells were harvested 36 to48 hours after transfection and lysates were assessed for TA expressionby Western Blot analysis (WB) as previously described (Chai 1999b). Theantibody used to detect TA was polyclonal rabbit antiserum generatedagainst a TA-maltose binding protein fusion protein (kindly provided byXandra Breakefield) at a 1:500 dilution. Additional antibodies used inthe experiments described here are the anti-HA mouse monoclonal antibody12CA5 (Roche) at 1:1,000 dilution, monoclonal mouse anti-GFP antibody(MBL) at 1:1,000 dilution, and for loading controls, anti α-tubulinmouse monoclonal antibody (Sigma) at 1:20,000 dilution.

Fluorescence visualization of fixed cells expressing GFP-tagged TA wasperformed with a Zeiss Axioplan fluorescence microscope. Nuclei werevisualized by staining with 5 μg/ml DAPI at room temperature for 10minutes. Digital images were collected on separate red, green and bluefluorescence channels using a Diagnostics SPOT digital camera. Live cellimages were collected with a Kodak MDS 290 digital camera mounted on anOlympus CK40 inverted microscope equipped for GFP fluorescence and phasecontrast microscopy. Digitized images were assembled using AdobePhotoshop 6.0.

Western Blot and Fluorescence Quantification. For quantification of WBsignal, blots were scanned with a Hewlett Packard ScanJet 5100C scanner.The pixel count and intensity of bands corresponding to TA and α-tubulinwere measured and the background signal subtracted using Scion Imagesoftware (Scion Corporation). Using the α-tubulin signal from controllanes as an internal reference, the TA signals were normalized based onthe amount of protein loaded per lane and the result was expressed aspercentage of TA signal in the control lane. Fluorescence quantificationwas determined by collecting three non-overlapping images per well atlow power (10×), and assessing the pixel count and intensity for eachimage with Bioquant Nova Prime software (BIOQUANT Image AnalysisCorporation). Background fluorescence, which was subtracted fromexperimental images, was determined by quantification of fluorescenceimages of untransfected cells at equivalent confluence, taken underidentical illumination and exposure settings.

Results

Expression of tagged TorsinA constructs. To test whether allele-specificsilencing could be applied to DYT1, a way to differentiate TAwt andTAmut proteins needed to be developed. Because TAwt and TAmut displayidentical mobility on gels and no isoform-specific antibodies areavailable, amino-terminal epitope-tagged TA constructs and GFP-TA fusionproteins were generated that would allow distinguishing TAwt and TAmut.The use of GFP-TA fusion proteins also facilitated the ability to screensiRNA suppression because it allowed visualization of TA levels inliving cells over time.

In transfected Cos-7 cells, epitope-tagged TA and GFP-TA fusion proteinexpression was confirmed by using the appropriate anti-epitope andanti-TA antibodies. Fluorescence microscopy in living cells showed thatGFP-TAwt and GFP-TAmut fusion proteins were expressed diffusely in thecell, primarily in the cytoplasm, although perinuclear inclusions werealso seen. It is important to note that these construct were designed toexpress reporter proteins in order to assess allele-specific RNAinterference rather than to study TA function. The N-terminal epitopeand GFP domains likely disrupt the normal signal peptide-mediatedtranslocation of TA into the lumen of the endoplasmic reticulum, whereTA is thought to function. Thus, while these constructs facilitatedexpression analysis in the studies described here, they are of limitedutility for studying TA function.

Silencing TorsinA with siRNA. Various siRNAs were designed to test thehypothesis that siRNA-mediated suppression of TA expression could beachieved in an allele-specific manner (FIG. 23). Because siRNA candisplay exquisite sequence specificity, the three base pair differencebetween mutant and wild type TOR1A alleles might be sufficient to permitthe design of siRNA that preferentially recognizes mRNA derived from themutant allele. Two siRNAs were initially designed to target TAmut(mutA-siRNA and mutB-siRNA) and one to target TAwt (wt-siRNA). Inaddition, a positive control siRNA was designed to silence both alleles(com-siRNA) and a negative control siRNA of irrelevant sequence(mis-siRNA) was designed. Cos-7 cells were first cotransfected withsiRNA and plasmids encoding either GFP-TAwt or untagged TAwt at a siRNAto plasmid ratio of 5:1. With wt-siRNA, potent silencing of TAwtexpression was observed to less than 1% of control levels, based onwestern blot analysis of cell lysates (FIGS. 24A and 24C). Withcom-siRNA, TAwt expression was suppressed to 30% of control levels. Incontrast, mutA-siRNA did not suppress TAwt and mutB-siRNA suppressedTAwt expression only modestly. These results demonstrate robustsuppression of TAwt expression by wild type-specific siRNA but notmutant-specific siRNA.

To assess suppression of TAmut, the same siRNAs were cotransfected withplasmids encoding untagged or HA-tagged TAmut. With mutA-siRNA ormutB-siRNA, marked, though somewhat variable, suppression of TAmutexpression was observed as assessed by western blot analysis of proteinlevels (FIGS. 24B and 24C). With com-siRNA, suppression of TAmutexpression was observed similar to what was observed with TAwtexpression. In contrast, wt-siRNA did not suppress expression of TAmut.Thus differential suppression of TAmut expression was observed byallele-specific siRNA in precisely the manner anticipated by theinventors.

To achieve even more robust silencing of TAmut, a third siRNA wasengineered to target TAmut (mutC-siRNA, FIG. 23). MutC-siRNA places theGAG deletion more centrally in the siRNA duplex. Because the centralportion of the antisense strand of siRNA guides mRNA cleavage, it wasreasoned that placing the GAG deletion more centrally might enhancespecific suppression of TAmut. As shown in FIG. 24, mutC-siRNAsuppressed TAmut expression more specifically and robustly than theother mut-siRNAs tested. In transfected cells, mutC-siRNA suppressedTAmut to less than 0.5% of control levels, and had no effect on theexpression of TAwt.

To confirm allele-specific suppression by wt-siRNA and mutC-siRNA,respectively, the inventors cotransfected cells with GFP-TAwt orGFP-TAmut together with mis-siRNA, wt-siRNA or mutC-siRNA. Levels of TAexpression were assessed 24 and 48 hours later by GFP fluorescence, andquantified the fluorescence signal from multiple images was quantified.The results (FIGS. 24D and 24E) confirmed the earlier western blotsresults in showing potent, specific silencing of TAwt and TAmut bywt-siRNA and mutC-siRNA, respectively, in cultured mammalian cells.

Allele-specific silencing in simulated heterozygous state. In DYT1, boththe mutant and wild type alleles are expressed. Once the efficacy ofsiRNA silencing was established, the inventors sought to confirm siRNAspecificity for the targeted allele in cells that mimic the heterozygousstate of DYT1. In plants and Caenorhabditis elegans, RNA-dependent RNApolymerase activity primed by introduction of exogenous RNA can resultin the spread of silencing signals along the entire length of thetargeted mRNA (Fire 1998, Tang 2003). No evidence for such a mechanismhas been discovered in mammalian cells (Schwarz 2002, Chiu 2002).Nonetheless it remained possible that silencing of the mutant allelemight activate cellular processes that would also inhibit expressionfrom the wild type allele. To address this possibility, Cos-7 cells werecotransfected with both GFP-TAwt and HA-TAmut, and suppression bymis-siRNA, wt-siRNA or mutC-siRNA was assessed. As shown in FIG. 25,potent and specific silencing of the targeted allele (either TAmut orTAwt) to levels less than 1% of controls was observed, with only slightsuppression in the levels of the non-targeted protein. Thus, in cellsexpressing mutant and wild type forms of the protein, siRNA can suppressTAmut while sparing expression of TAwt.

Discussion

In this study the inventors succeeded in generating siRNA thatspecifically and robustly suppresses mutant TA, the defective proteinresponsible for the most common form of primary generalized dystonia.The results have several implications for the treatment of DYT1dystonia. First and foremost, the suppression achieved was remarkablyallele-specific, even in cells simulating the heterozygous state. Inother words, efficient suppression of mutant TA occurred withoutsignificant reduction in wild type TA. Homozygous TA knockout mice dieshortly after birth, while the heterozygous mice are normal (Goodchild2002), suggesting an essential function for TA. Thus, therapy for DYT1needs to eliminate the dominant negative or dominant toxic properties ofthe mutant protein while sustaining expression of the normal allele inorder to prevent the deleterious consequences of loss of TA function.Selective siRNA-mediated suppression of the mutant allele fulfills thesecriteria without requiring detailed knowledge of the pathogenicmechanism.

An appealing feature of the present siRNA therapy is applicable to allindividuals afflicted with DYT1. Except for one unusual case (Leung2001, Doheny 2002, Klein 2002b), all persons with DYT1 have the same(GAG) deletion mutation (Ozelius 1997, Ozelius 1999). This obviates theneed to design individually tailored siRNAs. In addition, the fact thatthe DYT1 mutation results in a full three base pair difference from thewild type allele suggests that siRNA easily distinguishes mRNA derivedfrom normal and mutant TOR1A alleles.

It is important to recognize that DYT1 is not a fully penetrant disease(Fahn 1998, Klein 2002a). Even when expressed maximally, mutant TAcauses significant neurological dysfunction less than 50% of the time.Thus, even partial reduction of mutant TA levels might be sufficient tolower its pathological brain activity below a clinically detectablethreshold. In addition, the DYT1 mutation almost always manifests beforeage 25, suggesting that TAmut expression during a critical developmentalwindow is required for symptom onset. This raises the possibility thatsuppressing TAmut expression during development might be sufficient toprevent symptoms throughout life. Finally, unlike many other inheritedmovement disorders DYT1 is not characterized by progressiveneurodegeneration. The clinical phenotype must result primarily fromneuronal dysfunction rather than neuronal cell death (Hornykiewicz 1986,Walker 2002, Augood 2002, Augood 1999). This suggests the potentialreversibility of DYT1 by suppressing TAmut expression in overtlysymptomatic persons.

EXAMPLE 8 RNA Interference Improves Motor and NeuropathologicalAbnormalities in a Huntington's Disease Mouse Model

Huntington's disease (HD) is one of nine dominant neurodegenerativediseases resulting from polyglutamine repeat expansions (CAG codon, Q)in exon 1 of HD, leading to a toxic gain of function on the proteinhuntingtin (htt) (The Huntington's Disease Collaborative Research Group(1993) Cell 72, 971-83; Gusella et al, (2000) Nat Rev Neurosci 1,109-15). Hallmark HD characteristics include cognitive and behavioraldisturbance, involuntary movements (chorea), neuronal inclusions, andstriatal and cortical neurodegeneration (Gusella et al., (2000) Nat RevNeurosci 1, 109-15). Htt alleles containing greater than 35 CAG repeatsgenerally cause HD, with age-at-onset correlating inversely withexpansion length, a common characteristic of the polyglutamine repeatdisorders. The disease usually develops in mid-life, but juvenile-onsetcases can occur with CAG repeat lengths greater than 60. Death typicallyoccurs 10-15 years after symptom onset. Currently, no preventativetreatment exists for HD.

Therapies aimed at delaying disease progression have been tested in HDanimal models. For example, beneficial effects have been reported inanimals treated with substances that increase transcription ofneuroprotective genes (histone deacetylase) (Ferrante et al., (2003) JNeurosci 23, 9418-27); prevent apoptosis (caspase inhibitors) (Ona etal., (1999) Nature 399, 263-7); enhance energy metabolism (coenzymeQ/remacemide, creatine) (Ferrante et al., (2002) J Neurosci 22, 1592-9;Andreassen et al., (2001) Neurobiol Dis 8, 479-91); and inhibit theformation of polyglutamine aggregates (trehalose, Congo red, cystamine)(Tanaka et al., (2004) Nat Med 10, 148-54; Karpuj et al., (2002) Nat Med8, 143-9; Sanchez et al., (2003) Nature 421, 373-9). These approachestarget downstream and possibly indirect effects of disease alleleexpression. In contrast, no therapies have been described that directlyreduce mutant huntingtin gene expression, thereby targeting thefundamental, underlying pathological insult.

The therapeutic promise of silencing mutant htt expression wasdemonstrated in a tetracycline-regulated mouse model of HD (Yamamoto etal., (2000) Cell 101, 57-66). When mutant htt was inducibly expressed,pathological and behavioral features of the disease developed, includingthe characteristic neuronal inclusions and abnormal motor behavior. Uponrepression of transgene expression in affected mice, pathological andbehavioral features resolved. Thus, reduction of htt expression usingRNAi may allow protein clearance mechanisms within neurons to normalizemutant htt-induced changes. We hypothesize that directly inhibiting theexpression of mutant htt will slow or prevent HD-associated symptomonset in a relevant animal model.

Screening of putative therapies for HD has benefited from the existenceof several HD mouse models (Beal et al, (2004) Nat Rev Neurosci 5,373-84; Levine et al, (2004) Trends Neurosci 27, 691-7). HD-likephenotypes are displayed in knock-in mice (Lin et al., (2001) Hum MolGenet. 10, 137-44; Menalled et al., (2003) J Comp Neurol 465, 11-26),drug-induced models (McBride et al., (2004) J Comp Neurol 475, 211-9)and transgenic mice expressing full-length mutant huntingtin (e.g.YAC-transgenic mice) (Hodgson et al., (1999) Neuron 23, 181-92; Slow etal., (2003) Hum Mol Genet. 12, 1555-67; Reddy et al., (1998) Nat Genet.20, 198-202) or an N-terminal fragment of htt (Yamamoto et al., (2000)Cell 101, 57-66; Mangiarini et al., (1996) Cell 87(3), 493-506;Schilling et al., (1999) Hum Mol Genet. 8(3), 397-407). Mice expressingtruncated N-terminal fragments of huntingtin have been valuable forproof-of-principle evaluation of therapies because they show rapidlyprogressive motor abnormalities and striatal neuropathology, phenotypeswhich do not develop or develop very late in knock-in or YAC transgenicmice. Mice expressing truncated forms of huntingtin thus replicate moresevere forms of the disease. The present inventors tested if RNAinterference (RNAi) induced by short hairpin RNAs (sbRNAs) (Dykxhoom etal., (2003) Nat Rev Mol Cell Biol 4, 457-67) could reduce expression ofmutant htt and improve HD-associated abnormalities in a transgenic mousemodel of HD. It was found that RNAi directed against mutant humanhuntingtin (htt) reduced htt mRNA and protein expression in cell cultureand in HD mouse brain. It is important to note that htt gene silencingimproved behavioral and neuropathological abnormalities associated withHD.

Materials and Methods

Plasmids and Adeno-Associated Virus (AAV) construction. Myc-taggedHD-N171-82Q was expressed from a pCMV-HD-N171-82Q plasmid (Schilling etal., (1999) Hum Mol Genet. 8(3), 397-407). PCR (Pfu polymerase,Stratagene) was used to amplify the U6 promoter along with shRNAstargeting human huntingtin (shHD2.1; FIG. 26A), eGFP (shGFP) (Xia etal., (2002) Nat Biotechnol 20, 1006-1010); or E. coli β-galactosidase(bp 1152-1172; shLacZ). PCR products were cloned, verified by sequencingand inserted into pAAV.CMV.hrGFP, which contains AAV-2 ITRs, aCMV-hrGFP-SV40 polyA reporter cassette, and sequences used forhomologous recombination into baculovirus (Urabe et al., (2002) Hum GeneTher 13, 1935-1943). Recombinant AAV serotype 1 capsid vectors weregenerated as described (Urabe et al., (2002) Hum Gene Ther 13,1935-1943). AAV titers were determined by quantitative PCR and/or DNAslot blot and were 5×10¹² vector genomes/ml.

Animals. All animal studies were approved by the University of IowaAnimal Care and Use Committee. HD-N171-82Q mice were purchased fromJackson Laboratories, Inc. (Schilling et al., (1999) Hum Mol Genet.8(3), 397-407; Schilling et al., (2001) Neurobiol Dis 8, 405-18) andmaintained on a B6C3F1/J background. Heterozygous and age-matchedwildtype littermates were used for the experiments, as indicated.

Northern blots. HEK293 cells were transfected (Lipofectamine-2000;Invitrogen) with pCMV-HD-N171-82Q and plasmids expressing shHD2.1,shGFP, or shLacZ at shRNA:target ratios of 8:1. Forty-eight hourspost-transfection, RNA was harvested (Trizol Reagent; Invitrogen) and 10□g were assessed northern blot (NorthernMax; Ambion) using probes tohuman htt or human GAPDH. Band intensities were quantified using aphosphorimager (Storm 860 instrument and ImageQuant v1.2 software,Molecular Dynamics).

For in vivo studies, total RNA was isolated from hrGFP-positive striata.Thirty □g RNA was run on 15% polyacrylamide-urea gels, transferred toHybond N+ membranes (Amersham Pharmacia), then probed with ³²P-labeledsense oligonucleotides at 36° C. for 3 h, washed in 2×SSC (36° C.), andexposed to film.

Western blots. HEK293 cells were transfected as described with shHD2.1or shGFP singly or in combination with pCMV-HD-N171-82Q. Forty-eighthours later, cells were lysed to recover total protein. Western blotswere incubated with anti-myc (1:5,000; Invitrogen), anti full-lengthhuman htt (1:5,000; MAB2166; Chemicon), or anti-human β-actin (1:5,000;Clone AC-15; Sigma) followed by HRP-coupled goat anti-mouse or goatanti-rabbit secondary:antibodies (1:20,000 and 1:100,000, respectively;Jackson Immunochemicals). Blots were developed using ECL-Plus reagents(Amersham Biosciences). For evaluation of transduced brain, 3 week oldmice were injected as described and protein was harvested from striata 2weeks later. Twenty-five μg were run on SDS-PAGE gels as described,transferred to nitrocellulose, then probed with antibodies to detecthuman htt (1:500, mEM48; Gift from X. J. Li) and mouse prion protein(1:40,000; Chemicon International). Secondary antibody incubations wereperformed as described above.

Quantitative RT-PCR

In vitro shRNA dose response. HEK293 cells were transfected with 0(mock), 10, 100, or 1000 ng of shLacZ or shHD2.1 and RNA was harvested24 h later. Following DNase treatment (DNA-Free, Ambion), random-primed,first strand cDNA was generated from 500 ng total RNA (Taqman™ ReverseTranscription Reagents, Applied Biosystems) according to manufacturer'sprotocol. Taqman™ Assays were performed on an ABI Prism 7000 SequenceDetection System using Taqman™ 2× Universal PCR Master Mix (AppliedBiosystems) and Taqman™ primers/probe sets specific for human htt andmammalian rRNA (Applied Biosystems). Relative gene expression wasdetermined using the relative standard curve method.

In vivo huntingtin mRNA expression. Striata were dissected from 5.5month old mice, snap frozen in liquid nitrogen, and pulverized. cDNA wasgenerated as described above. Relative gene expression was assayed usingTaqman™ primers/probe sets specific for human htt and mammalian rRNA orAssays-By-Design Taqman™ primers/probes specific for mouse huntingtin(mHdh; Applied Biosystems). All values were calibrated to contralateral,uninjected striata. For human huntingtin detection; shHD2.1 samples, n=8striata; shLacZ, n=7; uninjected, n=4. For mouse Hdh detection; injectedHD samples, n=4; uninjected samples n=2.

AAV Injections

All animal procedures were pre-approved by the University of Iowa AnimalCare and Use Committee. AAV Injections were performed in 4 week old miceusing the following parameters (coordinates are reported with respect tothe bregma): Striatal: 0.5 mm anterior, 2.5 mm lateral, 2.5 mm depth, 5μl/site, 250 nl/min infusion rate. Cerebellar: 0.1 mm depth, 1 μl/site,250 nl/min infusion rate.

Behavioral Analysis

Stride length measurements. Mice injected bilaterally at 4 weeks of agewere analyzed at 4 months of age. Analyses were performed as describedpreviously (Carter et al., (1999) J Neurosci 19, 3248) with somemodifications. Specifically, mice were allowed to walk across apaper-lined chamber measuring 100 cm long, 10 cm wide, with 10 cm highwalls into an enclosed box. Mice were given one practice run and werethen tested three times to produce three separate footprint tracings,totaling 42 measurements each for front and rear footprints per mouse.Measurements were averaged and data presented as box plots. ANOVA withScheffe's post-hoc test was performed to determine statisticalsignificance. Uninjected mice, n=4; injected WT, n=3; injected N171-82Q,n=6 mice.

Rotarod performance test. Two separate experimental cohorts of mice wereinjected at 4 weeks of age and tested on the rotarod (Model 7650, UgoBasile Biological Research Apparatus) at 10 and 18 weeks of age aspreviously described (Xia et al., (2004) Nat Med 10, 816-820). Data fromtrials 2-4 for each day are presented as means ±S.E.M. Uninjected WT,n=6; shLacZ WT, n=5, shHD2.1 WT, n=6; uninjected N171-82Q, n=5; shLacZN171-82Q, n=10; shHD2.1 N171-82Q, n=11). Reported values are means±S.E.M.

Immunofluorescence

Forty □m free-floating coronal sections were stained with mEM48 antibody(1:500; 24 h, 4° C.), followed by Alexa-568 labeled goat anti-mousesecondary antibody (1:200; 4 h, room temp; Molecular Probes). Sectionswere mounted onto slides, covered in Gel/Mount (Biomeda Corp) and imageswere captured using fluorescent microscopy (Leica DM RBE or Zeissconfocal) equipped with a CCD-camera (SPOT RT, Diagnostics Instruments).Results shHD2.1 Reduces Human Huntingtin Expression In Vitro

In vitro screening was used to identify effective shRNAs directedagainst a CMV-promoter transcribed HD-N171-82Q mRNA, which is identicalto the pathogenic truncated huntingtin fragment transgene present inHD-N171-82Q mice (Schilling et al, (1999) Hum Mol Genet. 8(3), 397-407).Hairpin constructs targeting sequences in human exons 1-3 were evaluatedby co-transfection. One htt-targeted shRNA, shHD2.1 (FIG. 26A), reducedHD-N171-82Q mRNA and protein levels by ˜85 and ˜55% respectively,relative to control shRNA treated samples (FIG. 26B, C). Interestingly,none of the shRNAs tested that targeted exon 1 were functional underthese conditions and in this system. Additional siRNAs can be screenedas described herein to identify functional siRNAs targeting exon 1 ofthe HD gene.

To test if shHD2.1 could silence endogenous full-length human httexpression, HEK 293 cells were transfected with plasmids expressingshHD2.1 or shGFP. ShHD2.1, but not control shRNAs, directed genesilencing of endogenous htt mRNA and protein (FIGS. 26D, E). This systemcan be readily used to screen additional siRNAs targeting the HD gene.

Expression of shRNA in Mouse Brain

Next, the inventors tested U6 promoter-transcribed sbHD2.1 expression invivo and determined its effects on HD-associated symptoms in mice. Thispol III dependent promoter has not previously been evaluated in striatafor sustained expression in vivo, although shRNAs have been expressed inbrain using either the pol II-dependent CMV promoter in striatum (xia etal., (2002) Nat Biotechnol 20, 1006-1010) or the H1 promoter incerebellar degeneration models (Xia et al., (2004) Nat Med 10, 816-820).U6 promoter-driven shHD2.1, and the control hairpin shLacZ, were clonedinto adeno-associated virus (AAV) shuttle plasmids that contained aseparate CMV-humanized Renilla green fluorescent protein (hrGFP)reporter cassette (FIG. 27A). High-titer AAV1 particles (AAV.shHD2.1 andAAV.shLacZ), which have broad neuronal tropism, were generated (Urabe etal., (2002) Hum Gene Ther 13, 1935-1943), and hairpin expression wasassessed after injection into mouse striatum. The N171-82Q mouse modelwas used because shHD2.1 targets sequences in exon 2, precluding use ofthe R6/2 transgenic model, which expresses only exon 1 of the HD gene.As shown in FIG. 27B, precursor and processed shRNAs (˜50 nt and 21 nt,respectively) were expressed three weeks after transduction, indicatingsustained expression and appropriate processing of shRNAs in thestriatum. Analysis of coronal brain sections from injected mice showedwidespread transduction (FIG. 27C; hrGFP fluorescence) up to 5 monthspost-injection.

AAV.shHD2.1 Reduces HD-N171-82Q Expression In Vivo

The inventors next investigated the effects of RNAi on thecharacteristic HD-associated neuronal inclusions and HD-N171-82Q mRNAlevels in vivo. Tissues were harvested from end-stage HD-N171-82Q mice(˜5.5 months of age) because striatal inclusions are less robust atearlier ages in this model. In striata from HD-N171-82Q mice injectedwith AAV.shHD2.1, htt-reactive inclusions were absent in transducedcells compared to untransduced regions (FIG. 28A, lower panels; FIG.28B). Conversely, abundant inclusions were detected in transducedregions from AAV.shLacZ-injected HD mice (FIG. 28A, upper panels). Noinclusions were observed in WT mice (data not shown). In addition,western analysis revealed that soluble HD-N171-82Q monomer was decreasedin mouse striata transduced with AAV.shHD2.1 compared to uninjected orAAV.shLacZ-injected controls (FIG. 28C). The reduction in protein levelsdetected by immunohistochemistry and western blot was due to decreasedtransgene expression. HD-N171-82Q mRNA was reduced 51% to 55% inAAV.shHD2.1-injected HD mice relative to AAV.shLacZ-injected oruninjected HD mice (FIG. 28D). AAV.shHD2.1 and AAV.shLacZ had no effecton endogenous mouse htt expression (Avg. mHDH expression: Uninjected HD,1.00±0.09; Uninjected WT, 1.13±0.04; AAV.shLacZ injected HD, 1.10±0.08;AAV.shHD2.1 injected HD, 1.08±0.05).

Neuronal inclusions in HD-N171-82Q striata are variable. Inclusions maybe present in as few as 10% and up to 50% of all striatal neurons indifferent end-stage HD-N171-82Q mice (Schilling et al, (1999) Hum MolGenet. 8(3), 397-407). In contrast, robust and widespread EM48-positiveinclusions are present in cerebellar granule cells by ˜3 months of age[(Schilling et al., (1999) Hum Mol Genet. 8(3), 397-407) and FIG. 28],and cerebellar HD-N171-82Q mRNA levels are ˜8 fold higher relative tostriatum (QPCR, data not shown). This high-level cerebellar expressionis partially attributable to the transcriptional profile of the prionpromoter driving HD-N171-82Q transgene expression (Schilling et al,(1999) Hum Mol Genet. 8(3), 397-407). Cerebellar inclusions are nottypically found in brains of adult-onset HD patients. However,cerebellar pathology has been reported in juvenile onset HD cases, whichare the most severe forms of the disease, and interestingly, in Hdh140knock-in mice as early as 4 months of age (Menalled et al., (2003) JComp Neurol 465, 11-26; Nance et al., (2001) Ment Retard Dev Disabil ResRev 7, 153-7; Fennema—et al, (2004) Neurology 63, 989-95; Seneca et al,(2004) Eur J Pediatr.; Byers et al., (1973) Neurology 23, 561-9; Wheeleret al., (2002) Hum Mol Genet. 11, 633-40). The abundant inclusions inHD-N171-82Q cerebellar neurons provide a second target for assessing theeffects of AAV.shHD2.1 on target protein levels. Direct cerebellarinjections were done into a separate cohort of mice, and HD-N171-82Qexpression examined by immunofluorescence. Together the data show thatAAV.shHD2.1, but not control AAV.shLacZ, reduces mutant htt expressionand prevents formation of the disease-associated neuronal inclusions.

Striatal Delivery of AAV.shHD2.1 Improves Established BehavioralPhenotypes

The effects of shRNA treatment on established behavioral deficits andanimal weight were tested. RNAi directed to striatum did not normalizethe notable weight differences between HD-N171-82Q and WT mice(shHD2.1-injected, 22.7±3.8 g; shLacZ, 22.6±2.8 g; compared toage-matched wild-type mice (shHD2.1, 26.3±0.4; shLacZ, 27.3±5.8),confirming that intracerebral injection confines RNAi therapy to thesite of application (Schilling et al, (1999) Hum Mol Genet. 8(3),397-407; Xia et al., (2004) Nat Med 10, 816-820). However, significantimprovements in stride length measurements and rotarod deficits werenoted.

Stride length and rotarod tests were performed on uninjected mice, andmice injected bilaterally into striatum with AAVshHD2.1 or AAVshLacZ. Asshown in FIG. 29A, HD-N171-82Q mice display significantly shorter stridelengths than those of wild-type (WT) mice, consistent with prior work(Menalled et al., (2003) J Comp Neurol 465, 11-26; Carter et al., (1999)J Neurosci 19, 3248; Wheeler et al., (2002) Hum Mol Genet. 11, 633-40).Gait deficits in AAV.shHD2.1-treated HD-N171-82Q mice were significantlyimproved compared to AAV.shLacZ-treated (improvements for front and rearstrides, 13 and 15%, respectively; p<0.0001) and uninjected HD-N171-82Qmice (front and rear strides, 14 and 18%, respectively; p<0.0001). Gaitimprovements did not fully resolve, as all HD-N171-82Q groups remainedsignificantly different than their age-matched WT littermates. There wasno effect of AAV.shLacZ or AAV.shHD2.1 expression on stride lengths ofWT mice.

The accelerating rotarod test was used to confirm the beneficialbehavioral effects of RNAi targeted to the mutant human HD allele(Schilling et al., (1999) Hum Mol Genet. 8(3), 397-407). Mice were leftuninjected, or were injected bilaterally into the striatum withAAV.shLacZ or AAV.shHD2.1 at 4 weeks of age, followed by rotarodanalyses at 10- and 18-weeks of age (FIG. 29B). By 10 weeks, uninjectedand AAV.shLacZ-injected HD mice show impaired performance relative toall other groups, and continued to demonstrate significantly reducedperformance over the course of the study (p<0.05 relative to all othergroups). It is important to note that HD mice treated with AAVshHD2.1showed dramatic behavioral improvements relative to control-treated HDmice (p<0.0008) (FIG. 29B). AAV.shLacZ-treated HD mice showed a 22%decline (p<0.005; ANOVA), while AAV.shHD2.1-treated HD mice displayed amodest, non-significant 3% drop in rotarod performance between 10 and 18weeks of age. There was a partial normalization of rotarod deficits inHD mice injected with AAV.shHD2.1 compared to WT mice that wasconsistent with the gait analyses.

The inventors found no decline in stride length or rotarod performancebetween WT mice left untreated, or those injected with shRNA-expressingAAVs (FIG. 29A,B). However, at 10 weeks, there was a dramatic differencein rotarod performance between uninjected WT and all groups of injectedWT mice, which resolved by 18 weeks of age. These data suggest thatthere was some detrimental effect of direct brain injection on rotarodperformance from which the mice recovered over time. These data suggestthat RNAi expression in mammalian brain had no overt negative impact onmotor behavior (FIG. 29A,B).

Discussion

The inventors have shown that motor and neuropathological abnormalitiesin a relevant HD mouse model are significantly improved by reducingstriatal expression of a pathogenic huntingtin allele usingAAV1-delivered shRNA. The inventors have previously shown that RNAi canimprove neuropathology and behavioral deficits in a mouse model ofspino-cerebellar ataxia type 1 (SCA1) (Xia et al., (2004) Nat Med 10,816-820), a dominant neurodegenerative disorder that affects apopulation of neurons distinct from those degenerating in HD.

The shHD2.1 hairpin sequence reduced huntingtin expression in vitro andin vivo, and it is important to note, the present northern blot datasuggest that the processed active guide strand was protected by RISC invivo. The activity of the shRNAs could be improved using recentlydescribed rules for optimal shRNA design (Reynolds et al., (2004) NatBiotechnol 22, 326-30; Schwarz et al., (2003) Cell 115, 199-208;Khvorova et al., (2003) Cell 115, 505; Ui-Tei et al., (2004) NucleicAcids Res 32, 936-48).

Prior work demonstrated an essential role for huntingtin inembryogenesis and postnatal neurogenesis (Nasir et al, (1995) Cell 81,811-23; Duyao et al., (1995) Science 269, 407-10; White et al., (1997)Nat Genet. 17, 404-10; Dragatsis et al., (2000) Nat Genet. 26, 300-6).However the effect of partial reduction of normal huntingtin expressionin adult, post-mitotic neurons in vivo is unknown. In the current study,shHD2.1 reduced expression of a mutant, disease-causing human htttransgene, but had no effect on normal mouse huntingtin expression dueto sequence differences between mouse and human genes. In HD patients,shHD2.1 would be expected to reduce expression of both the mutant andnormal huntingtin alleles. The present data show that HD-like symptomscan be improved by even a partial reduction of mutant htt expression,suggesting that complete elimination of mutant allele expression may notbe required.

In summary, the inventors have shown that RNAi can dramatically improveHD-associated abnormalities, including pathological and behavioraldeficits, in a HD mouse model.

EXAMPLE 9 Huntington's Disease (HD)

Huntington's disease (HD) is one of several dominant neurodegenerativediseases that result from a similar toxic gain of function mutation inthe disease protein: expansion of a polyglutamine (polyQ)-encodingtract. It is well established that for HD and other polyglutaminediseases, the length of the expansion correlates inversely with age ofdisease onset. Animal models for HD have provided important clues as tohow mutant huntingtin (htt) induces pathogenesis. Currently, noneuroprotective treatment exists for HD. RNA interference has emerged asa leading candidate approach to reduce expression of disease genes bytargeting the encoding mRNA for degradation.

Short hairpin RNAs (shRNAs) were generated that significantly inhibitedhuman htt expression in cell lines. Importantly, the shRNAs weredesigned to target sequences present in HD transgenic mouse models. Thepresent studies test the efficacy of the shRNAs in HD mouse models bydetermining if inclusions and other pathological and behavioralcharacteristics that are representative of HD can be inhibited orreversed. In a transgenic model of inducible HD, pathology and behaviorimproved when mutant gene expression was turned off. These experimentsshow that RNAi can prevent or reverse disease.

Although the effect of partial reduction of wildtype htt in adultneurons is unknown, it is advantageous to target only mutant htt fordegradation, if possible. One polymorphism in linkage disequilibriumwith HD has been identified in the coding sequence for htt, and othersare currently being investigated. Disease allele-specific RNAi aredesigned using approaches that led to allele specific silencing forother neurogenetic disease models. This would allow directed silencingof the mutant, disease-causing expanded allele, leaving the normalallele intact.

Constitutive expression of shRNA can prevent the neuropathological andbehavioral phenotypes in a mouse model of Spinocerebellar Ataxia type I,a related polyQ disease. However, the constitutive expression of shRNAmay not be necessary, particularly for pathologies that take many yearsto develop but may be cleared in a few weeks or months. For this reason,and to reduce long-term effects that may arise if nonspecific silencingor activation of interferon responses is noted, controlled expressionmay be very important. In order to regulate RNAi for diseaseapplication, doxycycline-responsive vectors have been developed forcontrolled silencing in vitro.

HD researchers benefit from a wealth of animal models including sixtransgenic and four knock-in mouse models (Bates 2003). Expression isfrom the endogenous human promoter, and the CAG expansion in the R6lines ranges from 110 to approximately 150 CAGs. The R6/2 line is themost extensively studied line from this work. R6/2 mice show aggressivedegenerative disease, with age of symptom onset at 8-12 weeks, and deathoccurring at 10 to 13 weeks. Neuronal intranuclear inclusions, ahallmark of HD patient brain, appear in the striatum and cortex of theR6/2 mouse (Meade 2002).

Adding two additional exons to the transgene and restricting expressionvia the prion promoter led to an HD mouse model displaying important HDcharacteristics but with less aggressive disease progression (Shilling1999, Shilling 2001). The Borchelt model, N171-82Q, has greater thanwildtype levels of RNA, but reduced amounts of mutant protein relativeto endogenous htt. N171-82Q mice show normal development for the first1-2 months, followed by failure to gain weight, progressiveincoordination, hypokinesis and tremors. There are statisticallysignificant differences in the rotarod test, alterations in gait, andhindlimb clasping. Mice show neuritic pathology characteristic of humanHD. Unlike the Bates model, there is limited neuronal loss.

Detloff and colleagues created a mouse knock-in model with an extensionof the endogenous mouse CAG repeat to approximately 150 CAGs. Thismodel, the CHL2 line, shows more aggressive phenotypes than prior mouseknock-in models containing few repeats (Lin 2001). Measurableneurological deficits include clasping, gait abnormalities, nuclearinclusions and astrogliosis.

The present studies utilize the well-characterized Borchelt mouse model(N171-82Q, line 81), and the Detloff knock-in model, the CHL2 line. Theinitial targets for htt silencing were focused on sequences present inthe N171-82Q transgene (exons 1-3). The use of this model wasadvantageous in the preliminary shRNA development because the RNAisearch could focus on only the amino-terminal encoding sequences ratherthan the full length 14 kb mRNA. FIG. 31 depicts the one-step cloningapproach used to screen hairpins (Harper 2004). No effective shRNAs werefound in exon 1, but several designed against exon 2, denoted shHDEx2.1(5′-AAGAAAGAACTTTCAGCTACC-3′, SEQ ID NO:91), shHDEx2.2 19 nt(5′-AGAACTTTCAGCTACCAAG-3′ (SEQ ID NO:92)), or shHDEx2.2 21 nt5′-AAAGAACTTTCAGCTACCAAG-3′ (SEQ ID NO:93)) and exon 3 (shHDEx3.1 19 nt5′-TGCCTCAACAAAGTTATCA-3′ (SEQ ID NO:94) or shHDEx3.1 21 nt5′-AATGCCTCAACAAAGTTATCA-3′ (SEQ ID NO:95)) sequences were effective. Inco-transfection experiments with shRNA expressing plasmids and theN171-82Q transcript target, shHDEx2.1 reduced N171-Q82 transcript levelsby 80%, and protein expression by 60%.

In transient transfection assays shHDex2.1 did not silence a constructspanning exons 1-3 of mouse htt containing a 79 CAG repeat expansion,the mouse equivalent of N171-82Q. Next shHDEx2 into NIH 3T3 cells weretransfected to confirm that endogenous mouse htt, which is expressed inNIH 3T3 cells, would not be reduced. Surprisingly, shHDEx2.1 andshHDEx3.1 silenced full-length mouse htt. In contrast, shHDEx2.2silenced only the human N171-82Q transgene.

Yamamoto and colleagues and others have demonstrated that preformedinclusions can resolve (Yamamoto 2000). To test if RNAi could alsoreduce preformed aggregates, the inventors used a neuronal cell line,which, upon induction of Q80-eGFP expression, showed robust inclusionformation (Xia 2002). Cells laden with aggregates were mock-transduced,or transduced with recombinant virus expressing control shRNA, or shRNAsdirected against GFP. The inventors found dramatic reduction inaggregates as assessed by fluorescence. Quantification showed dosedependent effects (FIG. 32) that were corroborated by western blot (Xia2002).

As indicated in Example 1 above, viral vectors expressing siRNAs canmediate gene silencing in the CNS (Xia 2002). Also, these studies wereextended to the mouse model of spinocerebellar ataxia type 1 (SCA1). Thedata are important as they demonstrate that sbRNA is efficacious in theCNS of a mouse model of human neurodegenerative disease. The data alsosupport that sbRNA expression in brain is not detrimental to neuronalsurvival.

shRNAs can target the Exon 58 polymorphism. As described in Example 4above, a polymorphism in htt exon 58 is in linkage disequilibrium withHD (Ambrose 1994). Thirty eight percent of the HD population possesses a3-GAG repeat in exon 58, in contrast to the 4-GAG repeat found in 92% ofnon-HD patients. The polymorphism likely has no affect on htt, but itprovides a target for directing gene silencing to the disease allele. Asindicated in Example 4 above, in experiments to test if allele-specificsilencing for HD was possible, plasmids were generated that expressedshRNAs that were specific for the exon 58 polymorphism. The exon 583-GAG-targeting shRNAs were functional.

Developing vectors for control of RNAi in vivo. As demonstrated above,shRNA expressed from viral vectors is effective at directing genesilencing in brain. Also, viral vectors expressing shSCA1 inhibitedneurodegeneration in the SCA1 mouse model. ShRNA expression wasconstitutive in both instances. However, constitutive expression may notbe necessary, and could exacerbate any noted nonspecific effects. Thepresent inventors have developed and tested severaldoxycycline-regulated constructs. The construct depicted in FIG. 33showed strong suppression of target gene (GFP) expression after additionof doxycycline and RNAi induction.

RNAi can Protect, and/or Reverse, the Neuropathology in Mouse Models ofHuman Huntington's Disease

Two distinct but complimentary mouse models are used, the N171-82Qtransgenic and CHL2 knock-in mice. The former express a truncatedNH2-terminal fragment of human htt comprising exons 1-3 with an82Q-repeat expansion. The knock-in expresses a mutant mouse allele witha repeat size of ˜150. Neither shows significant striatal or corticalcell loss. Both therefore are suitable models for the early stages ofHD. They also possess similarities in mid- and end-stageneuropathological phenotypes including inclusions, gliosis, and motorand behavioral deficits that will permit comparison and validation. Onthe other hand, the differences inherent in the two models provideunique opportunities for addressing distinct questions regarding RNAitherapy. For example, N171-82Q transgenic mice have relatively earlydisease onset. Thus efficacy can be assessed within a few months, incontrast to 9 months or more in the CHL2 line. Because the data showedthat shHDEx2.2 targets the human transgene and not mouse HD, evaluatedisease-allele specific silencing in N171-82Q mice is evaluated. Incontrast, the CHL2 knock-in is important for testing how reducingexpression of both the mutant and wildtype alleles impacts on the HDphenotype. Finally, both models should be investigated because anytherapy for HD should be validated in two relevant disease models.

siRNA Against Human htt Protects Against Inclusion Formation in N171-82QMice

The data show that it is possible to silence the human N171-82Qtransgene in vitro, and work in reporter mice and SCA1 mouse modelsdemonstrated efficacy of RNAi in vivo in brain. shHDEx2.2 constructs,expressed from two vector systems with well-established efficacyprofiles in CNS, are now tested for their capacity to reduce mutanttransgenic allele expression in vivo. Further, the impact of shHDEx2.2on inclusion formation is assessed. Inclusions may not be pathogenicthemselves, but they are an important hallmark of HD and their presenceand abundance correlates with severity of disease in many studies.

Recombinant feline immunodeficiency virus (FIV) and adeno-associatedvirus (AAV) expressing shHDs are injected into N171-82Q. The levels ofshHDs expressed from FIV and AAV are evaluated, as is the ability toreduce htt mRNA and protein levels in brain, and subsequently affectinclusion formation.

Mice. N171-82Q mice developed by Borchelt and colleagues are used forthese experiments (Shilling 1999, Shilling 2001). The colony was set upfrom breeders purchased from Jackson Laboratories (N171-82Q, line 81)and are maintained as described (Shilling 1999, Shilling 2001). F1 pupsare genotyped by PCR off tail DNA, obtained when tagging weaned litters.

IC2 and EM48 have been used previously to evaluate N171-82Q transgeneexpression levels in brain by immuno-histochemistry (IHC) and westernblot (Zhou 2003, Trottier 1995). EM48 is an antibody raised against aGST-NH2 terminal fragment of htt that detects both ubiquitinated andnon-ubiquitinated htt-aggregates (Li 2000), and the IC2 antibodyrecognizes long polyglutamine tracts (Trottier 1995). By 4 weeksN171-82Q mice show diffuse EM48-positive staining in striata,hippocampus, cerebellar granule cells, and cortical layers IV and V(Shilling 1999, Shilling 2001). The present experiments focus on thestriatum and cortex because they are the major sites of pathology inhuman HD. TUNEL positivity and GFAP immunoreactivity are alsosignificant in striatal sections harvested from 3 month old N171-82Qmice (Yu 2003). At 4 months, punctate nuclear and cytoplasmicimmunoreactivity is also seen (Yu 2003).

Viruses. It is difficult to directly compare the two viruses under studyat equivalent doses; FIV is enveloped and can be concentrated andpurified, at best, to titers of 5×10⁸ infectious units/ml (iu/ml). FIVpseudotyped with the vesicular stomatitus glycoprotein (VSVg) are usedbecause of its tropism for neurons in the striatum (Brooks 2002). Incontrast, AAV is encapsidated and can be concentrated and purified totiters ranging from 1×10⁹ to 1×10¹¹ iu/ml, with 1×10¹⁰ titers onaverage. AAV serotype 5 is used because it is tropic for neurons instriatum and cortex, our target brain regions. Other serotypes of AAV,such as AAV-1 may also be used to neurons in striatum and cortex. Also,it diffuses widely from the injection site (Alisky 2000, Davidson 2000).Ten-fold dilutions of FIV and AAV generally results in a greater than10-fold drop in transduction efficiency, making comparisons at equaltiters, and dose escalation studies, unreasonable. Thus, both virusesare tested at the highest titers routinely available to get a fairassessment of their capacities for efficacy in N171-82Q mice. Allviruses express the humanized Renilla reniformis green fluorescentprotein (hrGFP) reporter transgene in addition to the shRNA sequence(FIG. 34). This provides the unique opportunity to look at individual,transduced cells, and to compare pathological improvements in transducedvs. untransduced cells.

Injections. Mice are placed into a David Kopf frame for injections. Miceare injected into the striatum (5 microliters; 100 nl/min) and thecortex (3 microliters; 75 nl/min) using a Hamilton syringe andprogrammable Harvard pump. The somatosensory cortex is targeted from aburr hole at −1.5 mm from Bregma, and 1.5 mm lateral. Depth is 0.5 mm.The striatum is targeted through a separate burr hole at +1.1 mm fromBregma, 1.5 mm lateral and 2 mm deep. Only the right side of the brainis injected, allowing the left hemisphere to be used as a control fortransgene expression levels and presence or absence of inclusions.

Briefly, groups of 4 week-old mice heterozygous for the N171-82Qtransgene and their age-matched wildtype littermates are injected withFIV (FIV groups are VSVg.FIV.shHDEx2.2, VSVg.FIVshlacZ, VSVg.hrGFP,saline) or AAV (AAV groups are AAV5.shHDEx2.2, AAV5shlacZ, AAV5 hrGFP,saline) (n=18/group; staggered injections because of the size of theexperiment). Names of shHDEx2.2 and shlacZ expressing viruses have beenshortened from shlacZ.hrGFP, for example, to make it easier to read, butall vectors express hrGFP as reporter. Nine mice/group are sacrificed at12 weeks of age to assess the extent of transduction (eGFP fluorescence;viral copy number/brain region), shRNA expression (northern for shRNAs,and inhibition of expression of the transgenic allele (QPCR and westernblot). The remaining groups are sacrificed at 5 months of age. Thisexperimental set up is repeated (to n=6/group) to confirm results andtest inter-experiment variability.

All mice in all groups are weighed bi-weekly (every other week) afterinitial weekly measurements. N171-82Q mice show normal weight gain up toapproximately 6 weeks, after which there are significant differenceswith their wildtype littermates.

PCR Analyses. Brains are harvested from mice sacrificed at 12 weeks ofage, and grossly evaluated for GFP expression to confirm transduction.The cortex and striatum from each hemisphere is dissected separately,snap frozen in liquid N2, pulverized with a mortar and pestle, andresuspended in Trizol (Gibco BRL). Separate aliquots are used forQ-RTPCR for N171-82Q transgenes and DNA PCR for viral genomes. Acoefficient of correlation is determined for transgene silencingrelative to viral genomes for both vector systems, for the regionsanalyzed and compared to contralateral striata and mice injected withcontrol vectors or saline.

The RNA harvested is used to evaluate activation ofinterferon-responsive genes. Bridges et al (Bridges 2003) and Sledz andcolleagues (Sledz 2003) found activation of 2′5′ oligo(A) polymerase(OAS) in cell culture with siRNAs and shRNAs, the latter expressed fromlentivirus vectors. Gene expression changes are assessed using QPCR forOAS, Stat1, interferon-inducible transmembrane proteins 1 and 2 andprotein kinase R (PKR). PKR activation is an initial trigger of thesignaling cascade of the interferon response.

Protein analyses. A second set of 3 brains/group are harvested forprotein analysis. Regions of brains are micro dissected as describedabove, and after pulverization are resuspended in extraction buffer (50mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1mM BetaME, 1× complete protease inhibitor cocktail) for analysis bywestern blot. HrGFP expression are evaluated and correlated todiminished levels of soluble N171-82Q using anti-GFP and antibodies tothe NH2-terminal region of htt (EM48) or the polyglutamine tract (IC2).

Histology. Histology is done on the remaining animals. Mice are perfusedwith 2% paraformaldehyde in PBS, brains blocked to remove thecerebellum, post-fixed ON, and then cryoprotected in 30% sucrose. Fullcoronal sections (40 μm) of the entire cerebrum are obtained using aMicrotome (American Products Co #860 equipped with a Super Histo Freezefreezing stage). Briefly, every section is collected, and sections 1-6are placed into 6 successive wells of a 24-well plate. Every 400microns, two sections each of 10 microns are collected for Nissl and H&Estaining. The process is repeated.

EM-48 immuno-staining reveals diffuse nuclear accumulations in N171-82Qmice as early as 4 weeks of age. In 6 mo. old mice inclusions areextensive (Shilling 2001). The increase in cytoplasmic and nuclear EM48immuno-reactivity, and in EM48 immuno-reactive inclusions over timeallow quantitative comparisons between transduced and untransducedcells. Again, control values are obtained from mice injected withshlacZ-expressing vectors, saline injected mice, and wt mice. Thecontralateral region is used as another control, with care taken to keepin mind the possibility of retrograde and anterograde transport of virusfrom the injection site.

Quantitation of nuclear inclusions is done using BioQuant™ software inconjunction with a Leitz DM RBE upright microscope equipped with amotorized stage (Applied Scientific Instruments). Briefly, floatingsections are stained with anti-NeuN (AMCA secondary) and EM48 antibodies(rhodamine secondary) followed by mounting onto slides. The regions tobe analyzed are outlined, and threshold levels for EM48 immunoreactivityset using sections from control injected mice. A minimum of 50hrGFP-positive and hrGFP negative neurons cells are evaluated per slide(5 slides/mouse), and inclusion intensity measured (arbitrary units).This is done for both striata and cortices. To quantitate cytoplasmicinclusions, the striatum is outlined and total EM48 aggregate densitymeasured. Threshold values are again done using control hemispheres andcontrol injected mice.

Additional wells of sections are stained with anti-GFAP,anti-neurofilament, and the lectin GSA to assay for viral orviral+hairpin induced gliosis, neuritic changes, and microglialactivation, respectively. GFAP-stained brain sections from N171-82Q miceshow gliosis by 4 months (Yu 1998), although earlier time points havenot been reported.

Stereology. In a separate experiment on N171-82Q mice and wt mice,unbiased stereology using BioQuant™ software is done to assesstransduction efficiency. Stereology allows for an unbiased assessment ofefficiency of transduction (number of cells transduced/input). AAV5(AAV5 hrGFP, AAV5shHD.hrGFP) and FIV (VSVg.FIVhrGFP, VSVg.FIVshHD.hrGFP)transduction efficiency is compared in the striatum and somatosensorycortex in HD and wildtype mice, with n=5 each. Mice are harvested at 12and 20 weeks. The cerebrum is sectioned in its entirety and stored at−20° C. until analysis. Briefly, six weeks after gene transfer withVSVg.FIVhrGFP (n=3) or AAV5 hrGFP (n=3), every section of an HD mousecerebrum is mounted and an initial assessment of the required numbers ofsections and grid and dissector size done using the coefficient of error(as determined by Martheron's quadratic approximation formula) as aguide.

The 171-82Q HD mouse model has important neuropathological andbehavioral characteristics relevant to HD. Onset of disease occursearlier than HD knock-in or YAC transgenic models, allowing an initial,important assessment of the protective effects of RNAi on thedevelopment of neuropathology and dysfunctional behavior, withoutincurring extensive long term housing costs. Admittedly, disease onsetis slower and less aggressive than the R6/2 mice created by Bates andcolleagues (Mangiarini 1996), but the R6/2 line is difficult to maintainand disease is so severe that it may be less applicable and lesspredicative of efficacy in clinical trials.

N171-82Q mice (n=6/group) and age-matched littermates (n=6/group) are beweighed twice a month from 4 wks on, and baseline rotarod testsperformed at 5 and 7 weeks of age. Numbers of mice per group are asdescribed in Schilling et al (Shilling 1999) in which statisticallysignificant differences between N171-82Q and wildtype littermates weredescribed. At 7 weeks of age (after testing is complete), AAV(AAVshHDEx2.2, AAVshlacZ, AAVhrGFP, saline) or FIV (FIVshEx2.2,FIVshlacZ, FIVhrGFP, saline) is injected bilaterally into the striatumand cortex. Rotarod tests are repeated at 3-week intervals starting atage 9 weeks, until sacrifice at 6 months. The clasping behavior isassessed monthly starting at 3 months.

Behavioral testing. N171-82Q mice are given four behavioral tests, allof which are standard assays for progressive disease in HD mouse models.The tests allow comparisons of behavioral changes resulting from RNAi tothose incurred in HD mouse models given other experimental therapies.For example, HD mice given cystamine or creatine therapy showed delayedimpairments in rotarod performance, and in some cases delayed weightloss (Ferrante 2000, Dedeoglu 2002, Dedeogu 2003) In addition to therotarod, which is used to assay for motor performance and generalneurological dysfunction, the activity monitor allows assessment of thedocumented progressive hypoactivity in N171-82Q mice. The beam analysisis a second test of motor performance that has also been used in HD micemodels (Carter 1999). Clasping, a phenotype of generalized neurologicaldysfunction, is straightforward and takes little time. Claspingphenotypes were corrected in R. Hen's transgenic mice possessing aninducible mutant htt.

Accelerated rotarod. N171-82Q and age-matched littermates are habituatedto the rotarod at week 4, and 4 trials per day for 4 days done on week 5and 7, and every 3 weeks hence using previously described assays(Shilling 1999, Clark 1997) in use in the lab. Briefly, 10 min trialsare run on an Economex rotarod (Columbus Instruments) set to acceleratefrom 4 to 40 rpm over the course of the assay. Latency to fall isrecorded and averages/group determined and plotted. Based on prior work(Shilling 1999) 6 mice will give sufficient power to assesssignificance.

Clasping behavior. Normal mice splay their limbs when suspended, butmice with neurological deficits can exhibit the opposite, with fore andhind limbs crunched into the abdomen (clasping). All mice are suspendedand scored for clasping monthly. The clasp must be maintained for atleast 30 sec. to be scored positive.

Activity monitor. Most HD models demonstrate hypokinetic behavior,particularly later in the disease process. This can be measured inseveral ways. One of the simplest methods is to monitor home cageactivity with an infrared sensor (AB-system 4.0, Neurosci Co., LTD).Measurements are taken over 3 days with one day prior habituation to thetesting cage (standard 12-hour light/dark cycle). Activity monitoring isdone at 12, 17, and 20 and 23 weeks of age.

Beam walking. N171Q-82Q and age matched littermates are assayed formotor performance and coordination using a series of successively moredifficult beams en route to an enclosed safety platform. The assay is asdescribed by Carter et al (Carter 1999). Briefly, 1 meter-length beamsof 28, 17 or 11 mm diameter are placed 50 cm above the bench surface. Asupport stand and the enclosed goal box flank the ends. Mice are trainedon the 11 mm beam at 6 weeks of age over 4 days, with 3 trials per day.If mice can traverse the beam in <20 sec. trials are initiated. A trialis then run on each beam, largest to smallest, with a 60 sec cutoff/beamand one minute rest between beams. A second trial is run and the meanscores of the two trials evaluated.

RNAi cannot replace neurons; it only has the potential to protectnon-diseased neurons, or inhibit further progression of disease at apoint prior to cell death. N171-82Q mice do not show noticeable cellularloss, and is therefore an excellent model of early HD in humans. Thegeneral methodology is the similar to that described above, except thatthe viruses are injected at 4 months, when N171-82Q mice have measurablebehavioral dysfunction and inclusions. Animals are sacrificed at endstage disease or at 8 months, whichever comes first. Histology, RNA andprotein in harvested brains are analyzed as described above.

It is important to confirm the biological effects of virally expressedshHDs in a second mouse model, as it is with any therapy. The Detloffknock-in mouse (the CHL2 line, also notated as HdhCAGQ150) is used as asecond model of early HD disease phenotypes. These mice have a CAGexpansion of approximately 150 units, causing brain pathologies similarto HD including gliosis and neural inclusions in the cortex andstriatum. They also show progressive motor dysfunction and otherbehavioral manifestations including rotarod deficits, clasping, gaitabnormalities and hypoactivity.

Heterozygous CHL2 mice express the mutant and wildtype allele at roughlyequivalent levels, and shRNAs directed against mouse HD silence bothtranscripts. shmHDEx2.1 causes reductions in gene expression, but notcomplete silencing. Disease severity in mouse models is dependent onmutant htt levels and CAG repeat length.

The inventors created shmHDEx2 (shRNA for murine HD) directed against aregion in mouse exon 2 that reduces expression of the full-length mouseHdh transcript in vitro. Transduction of neurons withshmHDEx2-expressing viruses, and its impacts on neuropathologicalprogression, behavioral dysfunction and the appearance of EM48immuno-reactive inclusions in CHL2 mice is tested. shmHD- orshlacZ-expressing vectors in CHL2 and wildtype brain is tested. In thisexperiment, virus is injected into the striatum of wt or CHL2 mice(10/group) using the coordinates described above, at 3 months of age.Two months later mice are sacrificed and brains removed and processedfor RNA (n=5/group) and protein (n=5).

A second study tests the vectors in the Detloff model. Briefly, 15 miceper group are injected into the striatum and cortex at 3 months of agewith AAV (AAVshmHD, AAVshlacZ, AAVhrGFP, saline) or FIV (VSVg.FIV.shmHD,VSVg.FIVshlacZ, VSVg.FIVhrGFP, saline) expressing the transgenesindicated. To assess the impact of RNAi, activity performed. The miceare sacrificed at 16-18 months of age and five brains/group areprocessed for histology and sections banked in 24-well tissue cultureplates. The remaining brains are processed for RNA (n=6) and proteinanalysis (n=5). Northern blots or western blots are required to analyzewildtype and mutant htt expression because the only distinguishingcharacteristic is size.

Development of Effective Allele-Specific siRNAs

Mutant htt leads to a toxic gain of function, and inhibiting expressionof the mutant allele has a profound impact on disease (Yamamoto 2000).Also, selectively targeting the disease allele would be desirable ifnon-disease allele silencing is deleterious. At the present time, thereis one documented disease linked polymorphism in exon 58 (Lin 2001).Most non-HD individuals have 4 GAGs in Hdh exon 58 while 38% of HDpatients have 3 GAGs. As described above, RNAi can be accomplishedagainst the 3-GAG repeat.

Prior work by the inventors showed the importance of using full-lengthtargets for testing putative shRNAs. In some cases, shRNAs would workagainst truncated, but not full-length targets, or vice-versa. Thus, itis imperative that testable, full-length constructs are made to confirmallele-specific silencing. The V5 and FLAG tags provide epitopes toevaluate silencing at the mRNA and protein levels. This is important asputative shRNAs may behave as miRNAs, leading to inhibition ofexpression but not message degradation.

Designing the siRNAs. Methods are known for designing siRNAs (Miller2003, Gonzalez-Alegre 2003, Xia 2002, Kao 2003). Information is alsoknow about the importance of maintaining flexibility at the 5′ end ofthe antisense strand for loading of the appropriate antisense sequenceinto the RISC complex (Khvorova 2003 Schwarz 2003). DNA sequences aregenerated by PCR. This method allows the rapid generation of manycandidate shRNAs, and it is significantly cheaper than buying shRNAs.Also, the inserts can be cloned readily into our vector shuttle plasmidsfor generation of virus. The reverse primer is a long oligonucleotideencoding the antisense sequence, the loop, the sense sequence, and aportion of the human U6 promoter. The forward primer is specific to thetemplate in the PCR reaction. PCR products are cloned directly intopTOPO blunt from InVitrogen, plasmids transformed into DH5a, andbacteria plated onto Kanr plates (the PCR template is Ampr). Kanr clonesare picked and sequenced. Sequencing is done with an extended ‘hotstart’ to allow effective read-through of the hairpin. Correct clonesare transfected into cells along with plasmids expressing the target orcontrol sequence (HttEx58.GAG3V5 and HttEx58.GAG4FLAG, respectively) andsilencing evaluated by western blot. Reductions in target mRNA levelsare assayed by Q-RTPCR. The control for western loading is neomycinphosphotransferase or hrGFP, which are expressed in thetarget-containing plasmids and provide excellent internal controls fortransfection efficiency. The control for Q-RTPCR is HPRT.

Cell lines expressing targets with the identified polymorphism orcontrol wildtype sequences are created. Target gene expression are undercontrol of an inducible promoter. PC6-3, Tet repressor (TetR+) cells, aPC-12 derivative with a uniform neuronal phenotype (Xia 2002) are used.PC6-3 cells are transfected with plasmids expressing HDEx58.GAG3V5(contains neo marker) and HDEx58GAG4FLG (contains puro marker), andG418+/puromycin+positive clones selected and characterized fortranscript levels and htt-V5 or htt-Flag protein levels.

FIV vectors expressing the allele specific shRNAs are generated and usedto test silencing in the inducible cell lines. FIV vectors infect mostepithelial and neuronal cell lines with high efficiency and aretherefore useful for this purpose. They also efficiently infect PC6-3cells. AAV vectors are currently less effective in in vitro screeningbecause of poor transduction efficiency in many cultured cell lines.

Cells are transduced with 1 to 50 infectious units/cell in 24-welldishes, 3 days after induction of mutant gene expression. Cells areharvested 72 h after infection and the effects on HDEx58.GAG3V5 orHDEx58GAG4FLG expression monitored.

EXAMPLE 10 Micro RNAi-Therapy for Polyglutamine Disease

Post-transcriptional gene silencing occurs when double stranded RNA(dsRNA) is introduced or naturally expressed in cells. RNA interference(RNAi) has been described in plants (quelling), nematodes, andDrosophila. This process serves at least two roles, one as an innatedefense mechanism, and another developmental (Waterhouse 2001 Fire 1999,Lau 2001, Lagos-Quintana 2001, Lee 2001). RNAi may regulatedevelopmental expression of genes via the processing of small,temporally expressed RNAs, also called microRNAs (Knight 2001, Grishok2001). Harnessing a cell's ability to respond specifically to smalldsRNAs for target mRNA degradation has been a major advance, allowingrapid evaluation of gene function (Gonczy 2000, Fire 1998, Kennerdell1998, Hannon 2002, Shi 2003, Sui 2002).

Most eukaryotes encode a substantial number of small noncoding RNAstermed micro RNAs (miRNAs) (Zeng 2003, Tijsterman 2004, Lee 2004, Pham2004). mir-30 is a 22-nucleotide human miRNA that can be naturallyprocessed from a longer transcript bearing the proposed miR-30 stem-loopprecursor. mir-30 can translationally inhibit an mRNA-bearing artificialtarget sites. The mir-30 precursor stem can be substituted with aheterologous stem, which can be processed to yield novel miRNAs and canblock the expression of endogenous mRNAs.

Huntington's disease (HD) and Spinocerebellar ataxia type I (SCA1) aretwo of a class of dominant, neurodegenerative diseases caused by apolyglutamine (polyQ) expansion. The mutation confers a toxic gain offunction to the protein, with polyQ length predictive of age of onsetand disease severity. There is no curative or preventative therapy forHD or SCA1, supporting the investigation of novel strategies. Asdescribed above, the inventors showed that gene silencing by RNAinterference (RNAi) can be achieved in vitro and in vivo by expressingshort hairpin RNAs (shRNAs) specific for mRNAs encoding ataxin-1 orhuntingtin. Currently, strong, constitutive po1III promoters (U6 and H1)are used to express shRNAs, which are subsequently processed intofunctional small interfering RNAs (siRNAs). However, strong,constitutive expression of shRNAs may be inappropriate for diseases thattake several decades to manifest. Moreover, high-level expression may beunnecessary for sustained benefit, and in some systems may induce anon-specific interferon response leading to global shut-down of geneexpression. The inventors therefore generated polII-expressed microRNAs(miRNAs) as siRNA shuttles as an alternative strategy. Due to theirendogenous nature, miRNA backbones may prevent the induction of theinterferon response.

Using human mir-30 as a template, miRNA shuttles were designed that uponprocessing by dicer released siRNAs specific for ataxin-1. Briefly, theconstructs were made by cloning a promoter (such as an induciblepromoter) and an miRNA shuttle containing an embedded siRNA specific fora target sequence (such as ataxin-1) into a viral vector. By cloning theconstruct into a viral vector, the construct can be effectivelyintroduced in vivo using the methods described in the Examples above.Constructs containing po1II-expressed miRNA shuttles with embeddedataxin-1-specific siRNAs were co-transfected into cells with GFP-taggedataxin-1, and gene silencing was assessed by fluorescence microscopy andwestern analysis. Dramatic arid dose-dependent gene silencing relativeto non-specific miRNAs carrying control siRNAs was observed. Thispo1II-based expression system exploits the structure of known miRNAs andsupports tissue-specific as well as inducible siRNA expression, andthus, serves as a unique and powerful alternative to dominantneurodegenerative disease therapy by RNAi.

Briefly, the constructs were made by cloning a promoter (such as aninducible promoter) and an miRNA shuttle containing an embedded siRNAspecific for a target sequence (such as ataxin-1) into a viral vector.By cloning the construct into a viral vector, the construct can beeffectively introduced in vivo using the methods described in theExamples above.

EXAMPLE 11 Huntington's Disease (HD)

Subsequent to the experiments described in Example 9 above, theinventors have made additional siRNA molecules specific for regions ofthe HD gene (FIG. 35). All of these sequences have been tested, and werefound to be effective in RNA interference.

shHD1.1=SEQ ID NO: 106

shHD1.2=SEQ ID NO: 107

shHD1.3=SEQ ID NO: 108

shHD1.4=SEQ ID NO: 109

shHD1.5=SEQ ID NO: 110

shHD1.6=SEQ ID NO: 111

shHD1.7=SEQ ID NO: 112

shHD1.8=SEQ ID NO: 113

shHD1.9=SEQ ID NO: 114

shHD2.1=SEQ ID NO: 115

shHD2.2=SEQ ID NO: 97

shHD2.3=SEQ ID NO: 116

shHD2.4=SEQ ID NO: 117

shHD2.5=SEQ ID NO: 118

shHD2.6=SEQ ID NO: 119

shHD3.1=SEQ ID NO: 120

shHD3.2=SEQ ID NO: 121

shHD4.1=SEQ ID NO: 122

shHD8.1=SEQ ID NO: 123

shHD8.2=SEQ ID NO: 124

shHD12.1=SEQ ID NO: 125

shHD17.1=SEQ ID NO: 126

shHD17.2=SEQ ID NO: 127

shHD22.1=SEQ ID NO: 128

shHD28.1=SEQ ID NO: 129

shHD30.1=SEQ ID NO: 130

shHD32.1=SEQ ID NO: 131

shHD34.1=SEQ ID NO: 132

shHD34.2=SEQ ID NO: 133

shHD35.1=SEQ ID NO: 134

shHD37.1=SEQ ID NO: 135

shHD38.1=SEQ ID NO: 136

shHD38.2=SEQ ID NO: 137

shHD40.1=SEQ ID NO: 138

shHD42.1=SEQ ID NO: 139

shHD42.2=SEQ ID NO: 140

shHD58.1=SEQ ID NO: 141

shHD58.2=SEQ ID NO: 90

shHD63.1=SEQ ID NO: 142

The normal human huntingtin gene is SEQ ID NO:143, and the correspondingnormal mouse huntingin gene is SEQ ID NO:144.

A particular nucleic acid sequence also encompasses variants. A variantof a molecule is a sequence that is substantially similar to thesequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.The sequences listed above also encompass nucleic acids containing knownanalogs of natural nucleotides that have similar binding properties asthe reference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. The present invention encompassesnucleic acid sequences wherein at least 12 of the nucleotides the sameas in the sequences provided, but wherein the remaining nucleotides maybe replaced with other nucleotides.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

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1. An isolated RNA duplex comprising a first strand of RNA and a secondstrand of RNA, wherein the first strand comprises at least 15 contiguousnucleotides encoded by shSCA1.F10 (SEQ ID NO:102), shSCA1.F11 (SEQ IDNO:103), SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, or SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:96 through SEQ ID NO:101 or SEQ ID NO:106through SEQ ID NO:142, and wherein the second strand is complementary toat least 12 contiguous nucleotides of the first strand.
 2. (canceled) 3.The RNA duplex of claim 1, wherein the duplex is between 19 and 25 basepairs in length.
 4. (canceled)
 5. The RNA duplex of claim 1, wherein thefirst and/or second strand further comprises a 3′ overhang region, a 5′overhang region, or both 3′ and 5′ overhang regions.
 6. The RNA duplexof claim 5, wherein the overhang region is from 1 to 10 nucleotides inlength.
 7. The RNA duplex of claim 1, wherein the first strand and thesecond strand are operably linked by means of an RNA loop strand to forma hairpin structure comprising a duplex structure and a loop structure.8. The RNA duplex of claim 7, wherein the loop structure contains from 4to 10 nucleotides.
 9. (canceled)
 10. The RNA duplex of claim 7, whereinthe loop structure corresponds to SEQ ID NO:61 or SEQ ID NO:64.
 11. Anexpression cassette comprising a nucleic acid encoding at least onestrand of the RNA duplex of claim
 1. 12. The expression cassette ofclaim 11, further comprising a promoter. 13-15. (canceled)
 16. Theexpression cassette of claim 11, wherein the expression cassette furthercomprises a polyadenylation signal. 17-18. (canceled)
 19. A vectorcomprising the expression cassette of claim
 11. 20. A vector comprisingtwo expression cassettes, a first expression cassette comprising anucleic acid encoding the first strand of the RNA duplex of claim 1 anda second expression cassette comprising a nucleic acid encoding thesecond strand of the RNA duplex of claim
 1. 21. A cell comprising thevector of claim
 19. 22-23. (canceled)
 24. The cell of claim 21, whereinthe vector is an adenoviral, lentiviral, adeno-associated viral (AAV),poliovirus, HSV, or murine Maloney-based viral vector. 25-33. (canceled)34. A viral vector comprising a promoter and an miRNA shuttle containingan embedded siRNA that specifically targets a target sequence associatedwith a condition amenable to siRNA therapy.
 35. (canceled)
 36. Thevector of claim 34, wherein the vector is an adenoviral, lentiviral,adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-basedviral vector.
 37. (canceled)
 38. The vector of claim 34, wherein thecondition amenable to siRNA therapy is a neurodegenerative disease.39-40. (canceled)
 41. The vector of claim 38, wherein the conditionamenable to siRNA therapy is Huntington's disease or a spinocerebellarataxia (SCA).
 42. (canceled)
 43. The vector of claim 34, wherein thetarget sequence is a sequence encoding ataxin-1 or huntingtin. 44-46.(canceled)
 47. An AAV-1 expressed siRNA comprising an isolated firststrand of RNA of 15 to 30 nucleotides in length and an isolated secondstrand of RNA of 15 to 30 nucleotides in length, wherein the first orsecond strand comprises a sequence that is complementary to a nucleotidesequence encoding a mutant Huntington's Disease protein, wherein atleast 12 nucleotides of the first and second strands are complementaryto each other and form a small interfering RNA (siRNA) duplex underphysiological conditions, and wherein the siRNA silences the expressionof the nucleotide sequence encoding the mutant Huntington's Diseaseprotein in the cell.
 48. The siRNA of claim 47, wherein the first and/orsecond strand further comprises a 3′ overhang region, a 5′ overhangregion, or both 3′ and 5′ overhang regions.
 49. (canceled)
 50. The siRNAof claim 47, wherein the first strand and the second strand are operablylinked by means of an RNA loop strand to form a hairpin structurecomprising a duplex structure and a loop structure.
 51. The siRNA ofclaim 50, wherein the loop structure contains from 4 to 10 nucleotides.52. The siRNA of claim 50, wherein the loop structure corresponds to SEQID NO:61 or SEQ ID NO:64.
 53. The siRNA of claim 47, wherein the firststrand corresponds to SEQ ID NO:60 and the second strand corresponds toSEQ ID NO:62.
 54. A vector comprising two expression cassettes, a firstexpression cassette comprising a nucleic acid encoding a first strand ofan RNA duplex corresponding to SEQ ID NO:60 and a second expressioncassette comprising a nucleic acid encoding a second strand of the RNAduplex corresponding to SEQ ID NO:62. 55-61. (canceled)